Modified therapeutic agents and compositions thereof

ABSTRACT

Methods and compositions are provided for extending the half-life of a therapeutic agent. One or more half-life extending moieties may be attached to a therapeutic agent, thereby extending the half life of the therapeutic agent. The modified therapeutic agents (mTAs) comprising one or more half-life extending moieties attached to a therapeutic agent may be used to treat a disease or condition in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage entry of InternationalApplication No. PCT/US2014/055457, filed Sep. 12, 2014, which claims thebenefit of U.S. Provisional Application No. 61/877,799 filed Sep. 13,2013, and U.S. Provisional Application No. 61/917,816 filed Dec. 18,2013, all of which are incorporated by reference herein in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 2, 2014, isnamed 41135-709-831-b ST25.txt and is 51,371 bytes in size.

BACKGROUND OF THE INVENTION

The development of therapeutic agents (e.g., biological drugs) is oftenhampered by short half-lives. The biological half-life or eliminationhalf-life of a substance is the time it takes for a substance (forexample a metabolite, drug, signaling molecule, radioactive nuclide, orother substance) to lose half of its pharmacologic, physiologic, orradiologic activity. As a result of the short half-life, patients areoften administered higher dosages more frequently, which may lead toreduced compliance, higher costs and greater risks of side effects.

Extended-release products are designed to prolong the absorption ofdrugs with short half-lives, thereby allowing longer dosing intervalswhile minimizing fluctuations in serum drug levels. Current strategiesused for extending half-lives are those that increase hydrodynamicvolume (PEGylation) or those that use FcRn-mediated recycling (albuminfusions). Attachment of polypeptides or lipophilic constituents to drugshas also been used to extend the half-life of a biological agent (U.S.Pat. Nos. 6,268,343; 5,750,497; 8,129,343).

The present disclosure provides modified therapeutic agents (mTAs) forimproving the biological, chemical, physiologic, pharmacologic,pharmacokinetic, and/or pharmacodynamic properties of a therapeuticagent.

SUMMARY OF THE INVENTION

Methods and compositions are provided for the formation of modifiedtherapeutic agents. The modified therapeutic agents (mTAs) may comprisea therapeutic agent and one or more half-life extending moieties,wherein the therapeutic agent is a peptide that is covalently attachedto each of the one or more half-life extending moieties via a cysteineresidue on the peptide; and the half-life of the modified therapeuticagent is longer than the half-life of the peptide alone. The mTAs maycomprise two or more half-life extending moieties. The two or morehalf-life extending moieties may be identical. The two or more half-lifeextending moieties may be different. A half-life extending moiety of theone or more half-life extending moieties may comprise a lipid, apolyglycol region, or a combination thereof. Each of the one or morehalf-life extending moieties may comprise a lipid. A half-life extendingmoiety of the one or more half-life extending moieties may comprise apolyglycol region. A half-life extending moiety of the one or morehalf-life extending moieties may comprise a lipid and a polyglycolregion. A half-life extending moiety of the one or more half-lifeextending moieties may comprise an extended recombinant polypeptide(XTEN) comprising (i) an amino acid sequence characterized in that thesum of glycine (G), alanine (A), serine (S), threonine (T), glutamate(E), aspartate (D), leucine (L) and proline (P) residues constitutesmore than about 70% of the total amino acid sequence; (ii) asubstantially non-repetitive amino acid sequence; (iii) an amino acidsequence that has less than about 10% alpha helices; and/or (iv) anamino acid sequence that has less than about 10% beta-sheets. The XTENmay comprise an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% thetotal amino acid sequence. The XTEN may comprise an amino acid sequenceselected from SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that is at least about 70% homologous to an amino acid sequenceselected from SEQ ID NOs: 66-67. The lipid may be selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof. The polyglycol region maycomprise one or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. Thepolyglycol region may comprise one thousand or more polyethylene glycolunits, polypropylene glycol units, or polybutylene glycol units, or acombination thereof. The peptide may comprise one or more amino acidadditions, deletions, or substitutions, or a combination thereof. Thepeptide may be selected from relaxin, H1 relaxin, H2 relaxin, H3relaxin, human INSL3, human INSL4, human INSL6, human IGF1, human IGFII,human insulin, oxyntomodulin, exenatide, exendin-4, glucagon-likeprotein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, aGLP-1R and GCGR dual agonist, leptin, betatrophin, FGF 21, GDF 11,ANGPTL3, peptide-based toxin, Moka, and VM-24, or a derivative thereof,the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. Thepeptide may be selected from relaxin, oxyntomodulin, exenatide,exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1Rand GIPR dual agonist, a GLP-1R and GCGR dual agonist, leptin,betatrophin, FGF 21, GDF 11, ANGPTL3, Toxin-550, Moka, and VM-24, or aderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof. The peptide may be relaxin or a derivative thereof, thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof. The peptide maybe encoded by an amino acid sequence comprising at least a portion of apolypeptide sequence selected from a group consisting of SEQ ID NO:10-56. The peptide may be encoded by an amino acid sequence comprising10 or more amino acids based on or derived from a polypeptide sequenceselected from a group consisting of SEQ ID NO: 10-56. The peptide maycomprise an amino acid sequence that is at least about 50% homologous toan amino acid sequence selected from the group comprising SEQ ID NO:10-56. The peptide may comprise an amino acid sequence that is at least80% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 10-56. The peptide may comprise one or more aminoacid sequences selected from the group comprising SEQ ID NO: 10-56. Thepeptide may comprise two or more amino acid sequences selected from thegroup comprising SEQ ID NO: 10-56. The cysteine residue may be locatedon the N-terminus or C-terminus of the peptide. The cysteine residue maybe located on a non-terminus position of the peptide. The cysteineresidue may be an amino acid addition or substitution on the peptide.The cysteine residue may be an amino acid addition or substitution on awild-type peptide.

Methods and compositions are provided for the formation of lipidconjugates (LCs). The lipid conjugates (LCs) may comprise (a) one ormore lipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,fatty amines, and fatty alcohols, and derivatives thereof; and (b) atherapeutic agent (TA), wherein the TA is a peptide and the one or morelipids are conjugated to the TA via an amino acid residue on thepeptide. The amino acid residue may be a cysteine or lysine. The aminoacid residue may be a cysteine. The amino acid residue may be an aminoacid addition or amino acid substitution on the peptide. The amino acidresidue may be located at the N-terminus or C-terminus of the peptide.The amino acid residue may be located at a non-terminus position of thepeptide. The TA may be a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. One ormore cysteine or lysine residues may be introduced by the one or moreamino acid additions or substitutions or a combination thereof. Thelipid conjugates (LCs) may comprise (a) one or more lipids, the lipidsselected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more lipids are conjugated to the one or more therapeutic agents viaa cysteine residue.

Further disclosed herein is a lipid conjugate (LC) comprising (a) one ormore lipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,fatty amines, and fatty alcohols, and derivatives thereof, and (b) atherapeutic agent (TA), wherein the TA comprises a relaxin peptide orderivative thereof and the one or more lipids are conjugated to the TAvia an amino acid residue on the peptide. Further disclosed herein is alipid conjugate (LC) comprising (a) one or more lipids, the lipidsselected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more TAs comprise a relaxin peptide, or derivative thereof, whereinthe one or more lipids are attached to the one or more therapeutic agentvia a cysteine residue.

Further disclosed herein is a lipid conjugate (LC) comprising one ormore lipids attached to one or more therapeutic agents (TAs), whereinthe one or more TAs comprise a modified relaxin peptide, and wherein themodified relaxin peptide comprises a wild-type relaxin polypeptide withone or more amino acid mutations. Further disclosed herein is a lipidconjugate (LC) comprising (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, fatty amines, and fattyalcohols, and derivatives thereof; and (b) a therapeutic agent (TA),wherein the TA comprises a modified relaxin peptide, and wherein themodified relaxin peptide comprises a wild-type relaxin polypeptide withone or more amino acid mutations.

Disclosed herein are pharmaceutical compositions comprising an LC,wherein the LC comprises (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols; and (b)one or more therapeutic agents (TAs), wherein the one or more lipids areconjugated to the one or more therapeutic agents via a cysteine residue.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, fatty amines, and fattyalcohols, and derivatives thereof; and (b) a therapeutic agent (TA),wherein the TA is a peptide and the one or more lipids are conjugated tothe TA via an amino acid residue on the peptide.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols; and (b)one or more therapeutic agents (TAs), wherein the one or more TAscomprise a relaxin peptide or derivative thereof, wherein the one ormore lipids are attached to the one or more therapeutic agent via acysteine residue.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, fatty amines, and fattyalcohols, and derivatives thereof; and (b) a therapeutic agent (TA),wherein the TA comprises a relaxin peptide or derivative thereof and theone or more lipids are conjugated to the TA via an amino acid residue onthe peptide.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises one or more lipids attached to one or moretherapeutic agents (TAs), wherein the one or more TAs comprise amodified relaxin peptide, and wherein the modified relaxin peptidecomprises a wild-type relaxin polypeptide with one or more amino acidmutations.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises one or more lipids attached to atherapeutic agent (TA), wherein the TA comprises a modified relaxinpeptide, and wherein the modified relaxin peptide comprises a wild-typerelaxin polypeptide with one or more amino acid mutations.

Disclosed herein is a method for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises (a) oneor more lipids, the lipids selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, fatty amines, and fatty alcohols, and derivatives thereof; and(b) a therapeutic agent (TA), wherein the TA is a peptide and the one ormore lipids are conjugated to the TA via an amino acid residue on thepeptide.

Further disclosed herein is a method for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises (a) oneor more lipids, the lipids selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, and fatty alcohols; and (b) one or more therapeutic agents(TAs), wherein the one or more lipids are conjugated to the one or moretherapeutic agents via a cysteine residue.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises (a) oneor more lipids, the lipids selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, and fatty alcohols; and (b) one or more therapeutic agents(TAs), wherein the one or more TAs comprise a relaxin peptide orderivative thereof, wherein the one or more lipids are attached to theone or more therapeutic agent via a cysteine residue.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises (a) oneor more lipids, the lipids selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, fatty amines, and fatty alcohols, and derivatives thereof; and(b) a therapeutic agent (TA), wherein the TA comprises a relaxin peptideor derivative thereof and the one or more lipids are conjugated to theTA via an amino acid residue on the peptide.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises one ormore lipids attached to one or more therapeutic agents (TAs), whereinthe one or more TAs comprise a modified relaxin peptide, and wherein themodified relaxin peptide comprises a wild-type relaxin polypeptide withone or more amino acid mutations.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises one ormore lipids attached to a therapeutic agent (TA) wherein the TAcomprises a modified relaxin peptide, and wherein the modified relaxinpeptide comprises a wild-type relaxin polypeptide with one or more aminoacid mutations.

Disclosed herein are kits comprising an LC, wherein the LC comprises (a)one or more lipids, the lipids selected from sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, fatty amines, and fatty alcohols, and derivativesthereof; and (b) a therapeutic agent (TA), wherein the TA is a peptideand the one or more lipids are conjugated to the TA via an amino acidresidue on the peptide.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises (a) one or more lipids, the lipids selected from sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or moretherapeutic agents (TAs), wherein the one or more lipids are conjugatedto the one or more therapeutic agents via a cysteine residue.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises (a) one or more lipids, the lipids selected from sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, fatty amines, and fatty alcohols, andderivatives thereof; and (b) a therapeutic agent (TA), wherein the TAcomprises a relaxin peptide or derivative thereof, and the one or morelipids are conjugated to the TA via an amino acid residue on thepeptide.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises (a) one or more lipids, the lipids selected from sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or moretherapeutic agents (TAs), wherein the one or more TAs comprise a relaxinpeptide or derivative thereof, and the one or more lipids are attachedto the one or more therapeutic agent via a cysteine residue.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises one or more lipids attached to a therapeutic agent (TA),wherein the TA comprises a modified relaxin peptide, and wherein themodified relaxin peptide comprises a wild-type relaxin polypeptide withone or more amino acid mutations.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises one or more lipids attached to one or more therapeutic agents(TAs), wherein the one or more TAs comprise a modified relaxin peptide,and wherein the modified relaxin peptide comprises a wild-type relaxinpolypeptide with one or more amino acid mutations.

Disclosed herein is a vector comprising a polynucleotide sequence of SEQID NO: 1-9.

Disclosed herein is a host cell comprising a polynucleotide sequence ofSEQ ID NO: 1-9.

Disclosed herein is a vector comprising a polynucleotide sequenceencoding a polypeptide of SEQ ID NO: 10-56.

Disclosed herein is a host cell comprising a polynucleotide sequenceencoding a polypeptide of SEQ ID NO: 10-56.

The LCs disclosed herein may further comprise one or morepolyethyleneglycol subunits.

The LCs disclosed herein may comprise one or more pegylated lipids.

The LC may have the structure:TA-A¹-P¹-L  Formula (I)wherein: TA is the therapeutic agent; A¹ is a chemical group linking TAand P¹ or L; P¹ is a bond or comprises polyglycol; and L is the lipid.P¹ may be a bond. A sulfur or nitrogen atom of an amino acid residue ofTA may be connected to A¹ via a chemical bond. P¹ may comprisepolyglycol. P¹ may be -PEG-A²-; wherein PEG is a chemical groupcomprising one or more polyethyleneglycol subunits; and A² is a chemicalgroup linking PEG and L. PEG may be selected from

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20. The one or more lipids may beselected from a group consisting of octadecanedioic acid,tetradecylamine, myristic acid, stearic acid, docosahexaenoic acid,lithocholic acid ester, cholic acid and palmitic acid.

In some embodiments described herein of an LC of Formula (I),

-   -   A¹ is selected from

and

-   -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, —NC(O)R⁵,        —NC(O)OR⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl,        (cycloalkyl)alkyl, or heteroalkyl;    -   k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   q is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments described herein of an LC of Formula (I),

-   -   A² is selected from a bond,

-   -   X is a bond, NR⁵, S, or 0;    -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl, or        heteroalkyl; R⁶ is OH or —NR⁵R⁵;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

The LC may have the structure:TA-A¹-P¹-L  Formula (Ia)wherein: TA is the therapeutic agent with a cysteine residue, whereinthe cysteine residue is connected to A¹; A¹ is a chemical group linkingTA and P¹; P¹ is a bond or -PEG-A²-; PEG is a chemical group comprisingone or more polyethyleneglycol subunits; A² is a chemical group linkingPEG and L; and L is the lipid.

The sulfur atom of the cysteine residue of the TA of Formula (Ia) may beconnected to A¹ via a chemical bond.

The PEG of an LC of Formula (Ia) may be selected from:

wherein

m and n may independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20.

In some embodiments described herein of an LC of Formula (Ia),

-   -   A¹ is selected from

and

-   -   R¹, R², R³, and R⁴ are independently selected from H, halo, CN,        —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;    -   p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   q is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments described herein of an LC of Formula (Ia),

-   -   A² is selected from a bond,

-   -   X is a bond, NR⁵, or 0;    -   R¹, R², R³, and R⁴ are independently selected from H, halo, CN,        —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.    -   P¹ may be PEG-A² for an LC of Formula (Ia).

The LC of Formula (I) or (Ia) may comprise a TA comprising a modifiedrelaxin peptide. The LC of Formula (I) or (Ia) may comprise one or morelipids selected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols.

Attachment of the one or more lipids to the therapeutic agent maycomprise covalent attachment. Attachment of the one or more lipids tothe modified relaxin peptide may comprise covalent attachment.

The one or more lipids may be attached to a therapeutic agent via acysteine residue. The one or more lipids may be attached to the modifiedrelaxin via a cysteine residue.

Disclosed herein is a compound having the structure of A³-P¹-L, wherein:A³ is a haloacetamide, maleimide, benzyl halide, or pyridyl disulfide;P¹ is a bond or -PEG-A²-; PEG is a chemical group comprising one or morepolyethyleneglycol subunits; A² is a chemical group linking PEG and L;and L is a lipid selected from sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols.

Further disclosed herein is a compound having the structure of A³-P¹-L,wherein: A³ is a haloacetamide, maleimide, benzyl halide, or pyridyldisulfide; P¹ is a bond or comprises polyglycol; and L is a lipidselected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols.

Disclosed herein are methods and compositions comprising one or morelipids. The one or more lipids may be selected from a group consistingof saturated fatty acids, unsaturated fatty acids, fatty di-acids, fattyamides, polyunsaturated fatty acids, short-chain fatty acids, mediumchain fatty acids, long chain fatty acid and very long chain fattyacids.

The lipids may be selected from a group consisting of propanoic acid,butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoicacid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid,tridecanoic acid, tetradecanoic acid, myristic acid, pentadecanoic acid,hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoicacid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoicacid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid,heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanoicacid, henatriacontanoic acid, dotriacontanoic acid, tritriacontanoicacid, tetratriacontanoic acid, pentatriacontanoic acid andhexatriacontanoic acid.

The one or more lipids may be selected from a group consisting ofmyristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidicacid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid,arachidonic acid, eicosapentanoic acid, erucic acid, docosahexaenoicacid.

The one or more lipids may be selected from a group consisting ofoctadecanedioic acid, tetradecylamine, myristic acid, stearic acid,docosahexaenoic acid, lithocholic acid ester, cholic acid and palmiticacid.

The one or more lipids may comprise myristic acid. The one or morelipids may comprise docosahexanoic acid. The one or more lipids maycomprise lithocholic acid ester. The one or more lipids may comprisecholic acid. The one or more lipids may comprise palmitic acid. The oneor more lipids may comprise octadecanedioic acid. The one or more lipidsmay comprise tetradecylamine. The one or more lipids may comprisestearic acid.

The fatty acids may comprise at least about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore carbon atoms.

The one or more lipids may comprise fatty alcohols derived from fattyacids selected from a group consisting of saturated fatty acids,unsaturated fatty acids, polyunsaturated fatty acids, short-chain fattyacids, medium chain fatty acids, long chain fatty acid and very longchain fatty acids.

The one or more lipids may be selected from a group consisting ofcholesterol, 7-OH cholesterol, 7,25-dihydroxycholesterol, cholic acid,chenodeoxycholic acid, lithocholic acid, deoxycholic acid, glycocholicacid, glycodeoxycholic acid, glycolithocholic acid, andglycochenodeoxycholic acid.

The one or more lipids may be selected from a group consisting of cholicacid, chenodeoxycholic acid, lithocholic acid, deoxycholic acid,glycocholic acid, glycodeoxycholic acid, glycolithocholic acid, andglycochenodeoxycholic acid.

The one or more lipids may be selected from a group consisting ofα-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol,β-tocotrienol, γ-tocotrienol and δ-tocotrienol.

Disclosed herein are methods and compositions comprising one or moretherapeutic agents (TAs). The methods and compositions may comprise atherapeutic agent (TA). The one or more TAs may comprise at least aportion of a protein, biomolecule, chemical, toxin, drug or anycombination thereof.

The one or more TAs may comprise at least a portion of a hormone,kinase, receptor, ligand, growth factor, regulatory protein, metabolicprotein, cytokine, antibody or any combination thereof.

The one or more TAs may comprise a peptide selected from relaxin, H1relaxin, H2 relaxin, H3 relaxin, human INSL3, human INSL4, human INSL6,human IGF1, human IGFII, human insulin, oxyntomodulin, exenatide,exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1Rand GIPR dual agonist, a GLP-1R and GCGR dual agonist, leptin,betatrophin, FGF 21, GDF 11, ANGPTL3, peptide-based toxin, Moka, andVM-24, and derivatives thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The one or more TAs maycomprise relaxin, oxyntomodulin, exenatide, exendin-4, glucagon-likeprotein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, aGLP-1R and GCGR dual agonist, leptin, betatrophin, FGF 21, GDF 11,ANGPTL3, peptide-based toxin, Moka, or VM-24, or a derivative thereof,the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. Theone or more TAs may comprise oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, or a GLP-1R and GCGR dual agonist, or a derivative thereof, thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof. The one or moreTAs may comprise H1 relaxin, H2 relaxin, H3 relaxin, human INSL3, humanINSL4, human INSL6, human IGF1, human IGFII, or human insulin, or aderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof. The one or more TAs may comprise a peptidyl toxin or aderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof. The peptidyl toxin or a derivative thereof may be refoldedusing a refolding buffer comprising ammonium sulfate.

The one or more TAs may comprise a relaxin peptide or derivativethereof, the derivative being a peptide comprising one or more aminoacid additions, deletions, or substitutions, or a combination thereof.The relaxin derivative may comprise a modified A-chain of relaxincomprising one or more amino acids that have been added, deleted, orsubstituted, or a combination thereof. The relaxin derivative maycomprise a modified B-chain of relaxin comprising one or more aminoacids that have been added, deleted, or substituted, or a combinationthereof. The relaxin derivative may comprise a modified A-chain ofrelaxin comprising one or more amino acids that have been added,deleted, or substituted, or a combination thereof; and a modifiedB-chain of relaxin comprising one or more amino acids that have beenadded, deleted, or substituted, or a combination thereof. The relaxinderivative may comprise a modified prorelaxin comprising one or moreamino acids that have been added, deleted, or substituted, or acombination thereof.

The TA may be oxyntomodulin or derivative thereof, the derivative beinga peptide comprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The TA may be exendin-4 orderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof. The TA may be exenatide or derivative thereof, the derivativebeing a peptide comprising one or more amino acid additions, deletions,or substitutions, or a combination thereof. The TA may be glucagon-likepeptide (GLP-1) or derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The TA may be glucagon orderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof.

The one or more TAs may be encoded by a nucleotide sequence selectedfrom a group consisting of SEQ ID NO: 1-9.

The one or more TAs may be encoded by a nucleotide sequence comprising20 or more nucleotides based on or derived from a nucleotide sequenceselected from a group consisting of SEQ ID NO: 1-9.

The one or more TAs may be encoded by a nucleotide sequence that is atleast about 50% homologous to a nucleotide sequence selected from agroup consisting of SEQ ID NO: 1-9.

The one or more TAs may be encoded by a nucleotide sequence that is atleast about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%homologous to a nucleotide sequence selected from a group consisting ofSEQ ID NO: 1-9.

The one or more TAs may be encoded by a nucleotide sequence comprising200 or fewer nucleotides.

The one or more TAs may be encoded by a nucleotide sequence comprising60-1500 or fewer nucleotides.

The one or more TAs may be encoded by an amino acid sequence comprisingat least a portion of a polypeptide sequence selected from a groupconsisting of SEQ ID NO: 10-56.

The one or more TAs are encoded by an amino acid sequence may comprise10 or more amino acids based on or derived from a polypeptide sequenceselected from a group consisting of SEQ ID NO: 10-56.

The one or more TAs may comprise an amino acid sequence comprising20-500 or more amino acids based on or derived from an amino acidsequence selected from a group consisting of SEQ ID NO: 10-56.

The one or more TAs may comprise an amino acid sequence that is at leastabout 50% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 10-56.

The one or more TAs may comprise an amino acid sequence that is at leastabout 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97% or 99%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 10-56.

The one or more TAs may comprise a relaxin peptide. The relaxin peptidemay comprise a modified relaxin peptide. The modified relaxin peptidemay comprise at least a portion of a wild-type relaxin peptidecomprising one or more amino acid mutations. The relaxin peptide maycomprise at least a portion of an A chain and/or B chain of a relaxinpeptide.

The relaxin peptide may comprise one or more amino acid mutations. Theone or more amino acid mutations may comprise a deletion, substitution,addition or a combination thereof. The one or more amino acid mutationsmay comprise addition of one or more amino acid residues to thewild-type relaxin polypeptide. The one or more amino acid mutations maycomprise substitution of one or more amino acid residues of thewild-type relaxin polypeptide. The one or more amino acid mutations maycomprise deletion of one or more amino acid residues of the wild-typerelaxin polypeptide.

The one or more amino acid mutations may comprise one or more amino acidsubstitutions of one or more amino acid residues in an A chain and/or Bchain of a wild-type relaxin peptide. The one or more amino acidmutations may comprise one or more amino acid substitutions of one ormore amino acid residues in an A chain of a wild-type relaxin peptide.The one or more amino acid substitutions of one or more amino acidresidues in the A chain may be selected from a group consisting of Y3C,A7C, T16C, R18C, S19C, or a combination thereof. The one or more aminoacid mutations may comprise one or more amino acid substitutions of oneor more amino acid residues in a B chain of a wild-type relaxin peptide.The one or more amino acid substitutions of one or more amino acidresidues in the B chain may be selected from a group consisting of S2C,M4C, S26C, and S29C, or any combination thereof. The one or more aminoacid mutations may comprise a Y3C substitution in an A chain of awild-type relaxin peptide. The one or more amino acid mutations maycomprise an A7C substitution in an A chain of a wild-type relaxinpeptide. The one or more amino acid mutations may comprise a T16Csubstitution in an A chain of a wild-type relaxin peptide. The one ormore amino acid mutations may comprise a R18C substitution in an A chainof a wild-type relaxin peptide. The one or more amino acid mutations maycomprise a S19C substitution in an A chain of a wild-type relaxinpeptide. The one or more amino acid mutations may comprise a S2Csubstitution in a B chain of a wild-type relaxin peptide. The one ormore amino acid mutations may comprise a M4C substitution in a B chainof a wild-type relaxin peptide. The one or more amino acid mutations maycomprise a S26C substitution in a B chain of a wild-type relaxinpeptide. The one or more amino acid mutations may comprise a S29Csubstitution in a B chain of a wild-type relaxin peptide.

The one or more amino acid mutations may comprise substituting one ormore amino acid residues of a wild-type relaxin peptide with a cysteineresidue. The one or more amino acid residues of the wild-type relaxinpeptide may be selected from a group consisting of alanine, methionine,arginine, serine, threonine, and tyrosine.

The one or more amino acid mutations may comprise adding one or moreamino acid residues to a wild-type relaxin peptide.

The one or more lipids for use in the methods and compositions disclosedherein may enhance one or more pharmacokinetic properties of the one ormore TAs.

The one or more lipids may enhance one or more pharmacokineticproperties of the one or more TAs by at least about 200% as measured bypharmacodynamics when compared to the one or more TAs not attached tothe one or more lipids. The one or more lipids may enhance one or morepharmokinetic properties of a TA by at least about 250% as measured bypharmacodynamics when compared to the TA not attached to the one or morelipids.

The one or more pharmacokinetic properties may comprise a half-life.

Disclosed herein are compounds having the structure of A³-P¹-L, wherein:A³ may be a haloacetamide, maleimide, benzyl halide, or pyridyldisulfide; P¹ may be a bond or -PEG-A²-; A² may be a chemical grouplinking PEG and L; L may be a lipid selected from sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols; and PEG may be selected from:

whereinm and n may be independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20.

In some embodiments described herein,

-   -   A² is selected from a bond,

-   -   X is a bond, NR⁵, or 0;    -   R¹, R², R³, and R⁴ are independently selected from H, halo, CN,        —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵; R⁵ is H,        alkyl, haloalkyl, arylalkyl, or heteroalkyl;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

Disclosed herein is a method of producing an LC of Formula (I), themethod comprising reacting the cysteine residue of TA with A³-P¹-L,wherein A³ is a reactive precursor to form A¹. The method of producingan LC of Formula (I), may comprise reacting the cysteine residue of TAwith A³-P¹-L, wherein A³ is haloacetamide, maleimide, benzyl halide, orpyridyl disulfide. A³ may be a haloacetamide. A³ may be abromoacetamide.

The pharmaceutical compositions disclosed herein may further compriseone or more pharmaceutically acceptable salts, excipients or vehicles.

The methods and compositions disclosed herein may be used to treat adisease or condition in a subject in need thereof. The disease orcondition may be a cardiovascular disorder. The disease or condition maybe acute heart failure. The disease or condition may be fibrosis. Thedisease or condition may be pain. The pain may be neuropathic pain orinflammatory pain.

The LCs disclosed herein may be administered with one or more additionaltherapeutic agents. The one or more additional therapeutic agents may beselected from a group consisting of an anti-inflammatory drug, a statin,a diuretic, a beta-blocker, an angiotensin converting enzyme inhibitor,an angiotensin II receptor blocker or any combination thereof. The oneor more additional therapeutic agents may be aspirin.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. The invention is best understood from the followingdetailed description when read in conjunction with the accompanyingdrawings. It is emphasized that, according to common practice, thevarious features of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1 shows a scheme for a chemical conjugation of pegylated lipidderivative to the L-cysteine modified relaxin, (boxed residues may bemutated to L-cysteine).

FIG. 2 shows relaxin modified with cysteine for lipid conjugation,(boxed residues may be mutated, for example to L-cysteine, lysine,glutamine, alanine).

FIG. 3 shows human relaxin 2 activating relaxin 2 receptor (RXFP2 orLGR7) in HEK293T cells expressing LGR7 and CRE-Luc reporter line.

FIG. 4. shows a schematic of a pVB008 relaxin2 linker vector andadditional schematics of other relaxin expression constructs. FIG. 4discloses “GGGRGG” as SEQ ID NO: 69.

FIG. 5. shows an agarose gel of the Relaxin2 S26C and Relaxin2 S29C PCRproducts

FIG. 6. shows an agarose gel of the Relaxin2 S26C linker and Relaxin2S29C linker PCR products.

FIG. 7. shows an agarose gel of restriction enzyme digested vectors(pVB008 and pAC145) and PCR products (Relaxin2, Relaxin2 S26C linker,Relaxin2 S29C linker, and Exenatide).

FIG. 8. shows a SDS-PAGE gel of relaxin and relaxin mutants.

FIG. 9. shows a SDS-PAGE gel of wild type relaxin with CBD tag at theN-terminus.

FIG. 10. shows (a) the disulfide bond pattern for the Toxin-550 peptide(wild-type), and (b) an exemplary in vitro folding pathway ofcysteine-knot Toxin-550 peptide, the middle structure depicting atwo-sulfide bond intermediate, in which a disulfide bond is formedbetween cysteine I and cysteine IV and between cysteine II and cysteineV.

FIG. 11 shows mouse pharmacokinetic data for a lipid conjugate.

FIG. 12 shows mouse pharmacokinetic data for wild-type relaxin.

FIG. 13 shows pubic ligament length data for a lipid conjugate atvarious timepoints.

FIG. 14 shows dose response data for a lipid conjugate at a 24-htimepoint.

FIG. 15 shows mouse pharmacokinetic data for a non-limiting example ofan XTEN-modified therapeutic agent (Relaxin-B-D1A,S29C-XTEN-288).

FIG. 16A-B show exemplary mTAs. Entries 1-10 depict exemplary mTAscomprising a relaxin A-chain and B-chain attached to a half-lifeextending moiety (represented by X). Entries 11-13 depict exemplary mTAscomprising toxin-550 attached to a half-life extending moiety(represented by X). X may be FA₁, FA₂, FA₃, FA₄, FA₅, FA₆, or FA_(Q), oranother lipid described herein. Disulfide bonds are depicted by bracketsconnecting two cysteine residues or by lines connecting two cysteineresidues. FIG. 16B discloses “GSCGG” and “GSGG” as SEQ ID NOS 72 and 71,respectively.

FIG. 17 shows an exemplary mTA. The exemplary mTA comprises a relaxinA-chain and B-chain attached to half-life extending moiety (representedby X). X may be XTEN-288 or XTEN-864. Disulfide bonds are depicted bybrackets connecting two cysteine residues or by lines connecting twocysteine residues.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are modified therapeutic agents (mTAs). Generally, themTAs may comprise a therapeutic agent (TA) and one or more half-lifeextending moieties, wherein the TA is attached to the one or morehalf-life extending moieties. The therapeutic agent may be a peptide.The TA may be covalently attached to each of the one or more half-lifeextending moieties. The TA may be attached to the one or more half-lifeextending moities via a cysteine residue on the therapeutic agent. Thehalf-life of the modified therapeutic agent may be longer than thehalf-life of the therapeutic agent alone. A half-life extending moietymay comprise a lipid, a polyglycol region, or a combination thereof. Ahalf-life extending moiety may comprise an extended recombinantpolypeptide (XTEN) comprising (i) an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 70% of the total amino acid sequence; (ii) asubstantially non-repetitive amino acid sequence; (iii) an amino acidsequence that has less than about 10% alpha helices; and/or (iv) anamino acid sequence that has less than about 10% beta-sheets. Ahalf-life extending moiety may be attached to the sulfur atom of acysteine residue on the therapeutic agent. Non-limiting examples of mTAsinclude lipid conjugates (LCs) and XTEN-modified therapeutic agents(XTEN-mTAs).

Disclosed herein are lipid conjugates (LCs). Generally, the LCs comprisea lipid and a therapeutic agent. The lipid conjugates (LCs) may comprise(a) one or more lipids, the lipids selected from sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, fatty amines, and fatty alcohols, and derivativesthereof and (b) a therapeutic agent (TA), wherein the therapeutic agentis a peptide and the one or more lipids are conjugated to thetherapeutic agent via an amino acid on the peptide.

The lipid conjugates (LCs) may comprise (a) one or more lipids, thelipids selected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more lipids are conjugated to the one or more therapeutic agents viaa cysteine residue.

Further disclosed herein is a lipid conjugate (LC) comprising (a) one ormore lipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols; and (b) one or more therapeutic agents (TAs),wherein the one or more TAs comprise a relaxin peptide or derivativethereof, wherein the one or more lipids are attached to the one or moretherapeutic agent via a cysteine residue.

Further disclosed herein is a lipid conjugate (LC) comprising one ormore lipids attached to one or more therapeutic agents (TAs), whereinthe one or more TAs comprise a modified relaxin peptide, and wherein themodified relaxin peptide comprises a wild-type relaxin polypeptide withone or more amino acid mutations. The LC may comprise one or more lipidsattached to a therapeutic agent comprising a modified relaxin peptide,wherein the modified relaxin peptide comprises a wild-type relaxinpolypeptide with one or more amino acid mutations.

Disclosed herein are pharmaceutical compositions comprising an LC,wherein the LC comprises (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, fatty amines, and fattyalcohols, and derivatives thereof; and (b) a therapeutic agent (TA),wherein the therapeutic agent is a peptide and the one or more lipidsare conjugated to the therapeutic agent via an amino acid on thepeptide.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols; and (b)one or more therapeutic agents (TAs), wherein the one or more lipids areconjugated to the one or more therapeutic agents via a cysteine residue.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols; and (b)one or more therapeutic agents (TAs), wherein the one or more TAscomprise a relaxin peptide or derivative thereof, wherein the one ormore lipids are attached to the one or more therapeutic agent via acysteine residue.

Further disclosed herein are pharmaceutical compositions comprising anLC, wherein the LC comprises one or more lipids attached to one or moretherapeutic agents (TAs), wherein the one or more TAs comprise amodified relaxin peptide, and wherein the modified relaxin peptidecomprises a wild-type relaxin polypeptide with one or more amino acidmutations. The pharmaceutical composition may comprise an LC, whereinthe LC comprises one or more lipids attached to a therapeutic agent(TA), wherein the TA comprises a modified relaxin peptide, and whereinthe modified relaxin peptide comprises a wild-type relaxin polypeptidewith one or more amino acid mutations.

Disclosed herein is a method for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject a composition comprising a an LC, wherein the LC comprises (a)one or more lipids, the lipids selected from sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, fatty amines, and fatty alcohols, and derivativesthereof; and (b) a therapeutic agent (TA), wherein the therapeutic agentis a peptide and the one or more lipids are conjugated to thetherapeutic agent via an amino acid on the peptide.

Further disclosed herein is a method for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising a an LC, wherein the LC comprises (a)one or more lipids, the lipids selected from sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols; and (b) one or more therapeuticagents (TAs), wherein the one or more lipids are conjugated to the oneor more therapeutic agents via a cysteine residue.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises (a) oneor more lipids, the lipids selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, and fatty alcohols; and (b) one or more therapeutic agents(TAs), wherein the one or more TAs comprise a relaxin peptide orderivative thereof, wherein the one or more lipids are attached to theone or more therapeutic agent via a cysteine residue.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises one ormore lipids attached to one or more therapeutic agents (TAs), whereinthe one or more TAs comprise a modified relaxin peptide, and wherein themodified relaxin peptide comprises a wild-type relaxin polypeptide withone or more amino acid mutations. The method of treating a disease orcondition in a subject in need thereof may comprise administering to thesubject a composition comprising an LC, wherein the LC comprises one ormore lipids attached to a therapeutic agent (TA), wherein the TAcomprises a modified relaxin peptide, and wherein the modified relaxinpeptide comprises a wild-type relaxin polypeptide with one or more aminoacid mutations.

Disclosed herein are kits comprising an LC, wherein the LC comprises (a)one or more lipids, the lipids selected from sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, fatty amines, and fatty alcohols, and derivativesthereof; and (b) a therapeutic agent (TA), wherein the therapeutic agentis a peptide and the one or more lipids are conjugated to thetherapeutic agent via an amino acid on the peptide.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises (a) one or more lipids, the lipids selected from sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or moretherapeutic agents (TAs), wherein the one or more lipids are conjugatedto the one or more therapeutic agents via a cysteine residue.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises (a) one or more lipids, the lipids selected from sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or moretherapeutic agents (TAs), wherein the one or more TAs comprise a relaxinpeptide or derivative thereof, wherein the one or more lipids areattached to the one or more therapeutic agent via a cysteine residue.

Further disclosed herein are kits comprising an LC, wherein the LCcomprises one or more lipids attached to one or more therapeutic agents(TAs), wherein the one or more TAs comprise a modified relaxin peptide,and wherein the modified relaxin peptide comprises a wild-type relaxinpolypeptide with one or more amino acid mutations. The kit may comprisean LC, wherein the LC comprises one or more lipids attached atherapeutic agent (TA), wherein the TA comprises a modified relaxinpeptide, and wherein the modified relaxin peptide comprises a wild-typerelaxin polypeptide with one or more amino acid mutations.

Further disclosed herein are LCs having the structure:TA-A¹-P¹-L  Formula (I)wherein: TA is the therapeutic agent; A¹ is a chemical group linking TAand P¹ or L; P¹ is a bond or comprises polyglycol; and L is the lipid.

Further disclosed herein are LCs having the structure:TA-A¹-P¹-L  Formula (Ia)wherein: TA is the therapeutic agent with a cysteine residue, whereinthe cysteine residue is connected to A¹; A¹ is a chemical group linkingTA and P¹; P¹ is a bond or -PEG-A²-; PEG is a chemical group comprisingone or more polyethyleneglycol subunits; A² is a chemical group linkingPEG and L; and L is the lipid.

Disclosed herein are compounds having the structure of A³-P¹-L, wherein:A³ may be a haloacetamide, maleimide, benzyl halide, or pyridyldisulfide; P¹ may be a bond or -PEG-A²-; A² may be a chemical grouplinking PEG and L; L may be a lipid selected from sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols; and PEG may be selected from:

whereinm and n may be independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20.

Disclosed herein is a method of producing an LC of Formula (Ia), themethod comprising reacting the cysteine residue of TA with A³-P¹-L,wherein A³ is a reactive precursor to form A¹. The method of producingan LC of Formula (Ia) may comprise reacting the cysteine residue of TAwith A³-P¹-L, wherein A³ is haloacetamide, maleimide, benzyl halide, orpyridyl disulfide. A³ may be a haloacetamide. A³ may be abromoacetamide.

Before the present methods, kits and compositions are described ingreater detail, it is to be understood that this invention is notlimited to particular method, kit or composition described, as such may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the present inventionwill be limited only by the appended claims. Examples are put forth soas to provide those of ordinary skill in the art with a completedisclosure and description of how to make and use the present invention,and are not intended to limit the scope of what the inventors regard astheir invention nor are they intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Methods and compositions are provided for producing mTAs that extend thehalf-life of a therapeutic agent. These methods and compositions findtherapeutic use in a number of diseases, for example, cardiovasculardisease may be more effectively treated with a half-life extensionmolecule conjugated to relaxin than by relaxin alone. These and otherobjects, advantages, and features of the invention will become apparentto those persons skilled in the art upon reading the details of thecompositions and methods as more fully described below.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Modified Therapeutic Agent (mTA)

Disclosed herein are modified therapeutic agents (mTAs) may comprise atherapeutic agent (TA) and one or more half-life extending moieties,wherein the TA is attached to the one or more half-life extendingmoieties. The therapeutic agent may be a peptide. The TA may becovalently attached to each of the one or more half-life extendingmoieties. The TA may be attached to the one or more half-life extendingmoities via a cysteine residue on the therapeutic agent. The half-lifeof the modified therapeutic agent may be longer than the half-life ofthe therapeutic agent alone. A half-life extending moiety may comprise alipid, a polyglycol region, or a combination thereof. A half-lifeextending moiety may comprise an extended recombinant polypeptide (XTEN)comprising (i) an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 70% of the total amino acid sequence; (ii) a substantiallynon-repetitive amino acid sequence; (iii) an amino acid sequence thathas less than about 10% alpha helices; and/or (iv) an amino acidsequence that has less than about 10% beta-sheets. A half-life extendingmoiety may be attached to the sulfur atom of a cysteine residue on thetherapeutic agent. Non-limiting examples of mTAs include lipidconjugates (LCs) and XTEN-modified therapeutic agents (XTEN-mTAs).

Disclosed herein are modified therapeutic agents (mTAs) comprising atherapeutic agent and one or more half-life extending moieties, whereinthe therapeutic agent is a peptide that is covalently attached to eachof the one or more half-life extending moieties via a cysteine residueon the peptide; and the half-life of the modified therapeutic agent islonger than the half-life of the peptide alone. The peptide may compriseone or more amino acid additions, deletions, substitutions, or acombination thereof. The cysteine residue may be an amino acid additionor substitution on the peptide. The cysteine residue may be an aminoacid addition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid; and the half-life of the modifiedtherapeutic agent is longer than the half-life of the peptide alone. Thepeptide may comprise one or more amino acid additions, deletions,substitutions, or a combination thereof. The cysteine residue may be anamino acid addition or substitution on the peptide. The cysteine residuemay be an amino acid addition or substitution on a wild-type peptide.The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid; the peptide is selected from relaxin, H1relaxin, H2 relaxin, H3 relaxin, human INSL3, human INSL4, human INSL6,human IGF1, human IGFII, human insulin, oxyntomodulin, exenatide,exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1Rand GIPR dual agonist, a GLP-1R and GCGR dual agonist, leptin,betatrophin, FGF 21, GDF 11, ANGPTL3, peptide-based toxin, Moka, andVM-24, or a derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide. The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid; the peptide is selected from relaxin,oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1 (GLP-1),GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, leptin, betatrophin, FGF 21, GDF 11, ANGPTL3, Toxin-550, Moka,and VM-24, or a derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide. The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid; the peptide is relaxin or a derivativethereof, the derivative being a peptide comprising one or more aminoacid additions, deletions, or substitutions, or a combination thereofand the half-life of the modified therapeutic agent is longer than thehalf-life of the peptide alone. The cysteine residue may be an aminoacid addition or substitution on the peptide. The cysteine residue maybe an amino acid addition or substitution on a wild-type peptide. ThemTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid; the therapeutic agent is encoded by an aminoacid sequence comprising at least a portion of a polypeptide sequenceselected from a group consisting of SEQ ID NO: 10-56; and the half-lifeof the modified therapeutic agent is longer than the half-life of thepeptide alone. The peptide may comprise one or more amino acidadditions, deletions, substitutions, or a combination thereof. Thecysteine residue may be an amino acid addition or substitution on thepeptide. The cysteine residue may be an amino acid addition orsubstitution on a wild-type peptide. The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, fatty amines, and fatty alcohols, and derivatives thereof andthe half-life of the modified therapeutic agent is longer than thehalf-life of the peptide alone. The peptide may comprise one or moreamino acid additions, deletions, substitutions, or a combinationthereof. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, fatty amines, and fatty alcohols, and derivatives thereof thepeptide is selected from relaxin, H1 relaxin, H2 relaxin, H3 relaxin,human INSL3, human INSL4, human INSL6, human IGF1, human IGFII, humaninsulin, oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1(GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R andGCGR dual agonist, leptin, betatrophin, FGF 21, GDF 11, ANGPTL3,peptide-based toxin, Moka, and VM-24, or a derivative thereof, thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof; and the half-lifeof the modified therapeutic agent is longer than the half-life of thepeptide alone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, fatty amines, and fatty alcohols, and derivatives thereof; thepeptide is selected from relaxin, oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, leptin, betatrophin, FGF 21,GDF 11, ANGPTL3, Toxin-550, Moka, and VM-24, or a derivative thereof,the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof; andthe half-life of the modified therapeutic agent is longer than thehalf-life of the peptide alone. The cysteine residue may be an aminoacid addition or substitution on the peptide. The cysteine residue maybe an amino acid addition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, fatty amines, and fatty alcohols, and derivatives thereof; thepeptide is relaxin or a derivative thereof, the derivative being apeptide comprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region; and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The peptide may comprise one or more amino acid additions,deletions, substitutions, or a combination thereof. The cysteine residuemay be an amino acid addition or substitution on the peptide. Thecysteine residue may be an amino acid addition or substitution on awild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region; the peptide is selected fromrelaxin, H1 relaxin, H2 relaxin, H3 relaxin, human INSL3, human INSL4,human INSL6, human IGF1, human IGFII, human insulin, oxyntomodulin,exenatide, exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon,a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, leptin,betatrophin, FGF 21, GDF 11, ANGPTL3, peptide-based toxin, Moka, andVM-24, or a derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region; the peptide is selected fromrelaxin, oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1(GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R andGCGR dual agonist, leptin, betatrophin, FGF 21, GDF 11, ANGPTL3,Toxin-550, Moka, and VM-24, or a derivative thereof, the derivativebeing a peptide comprising one or more amino acid additions, deletions,or substitutions, or a combination thereof and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region; the peptide is relaxin or aderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof and the half-life of the modified therapeutic agent is longerthan the half-life of the peptide alone. The cysteine residue may be anamino acid addition or substitution on the peptide. The cysteine residuemay be an amino acid addition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region; the therapeutic agent is encodedby an amino acid sequence comprising at least a portion of a polypeptidesequence selected from a group consisting of SEQ ID NO: 10-56; and thehalf-life of the modified therapeutic agent is longer than the half-lifeof the peptide alone. The cysteine residue may be an amino acid additionor substitution on the peptide. The cysteine residue may be an aminoacid addition or substitution on a wild-type peptide.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a lipid and a polyglycol region; and the half-life ofthe modified therapeutic agent is longer than the half-life of thepeptide alone. The peptide may comprise one or more amino acidadditions, deletions, substitutions, or a combination thereof. Thecysteine residue may be an amino acid addition or substitution on thepeptide. The cysteine residue may be an amino acid addition orsubstitution on a wild-type peptide. The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region and a lipid selected fromsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof and the half-life of the modified therapeuticagent is longer than the half-life of the peptide alone. The peptide maycomprise one or more amino acid additions, deletions, substitutions, ora combination thereof. The cysteine residue may be an amino acidaddition or substitution on the peptide. The cysteine residue may be anamino acid addition or substitution on a wild-type peptide. The mTA maybe an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region and a lipid selected fromsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof the peptide is selected from relaxin, H1relaxin, H2 relaxin, H3 relaxin, human INSL3, human INSL4, human INSL6,human IGF1, human IGFII, human insulin, oxyntomodulin, exenatide,exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1Rand GIPR dual agonist, a GLP-1R and GCGR dual agonist, leptin,betatrophin, FGF 21, GDF 11, ANGPTL3, peptide-based toxin, Moka, andVM-24, or a derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide. The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region and a lipid selected fromsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof the peptide is selected from relaxin,oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1 (GLP-1),GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, leptin, betatrophin, FGF 21, GDF 11, ANGPTL3, Toxin-550, Moka,and VM-24, or a derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide. The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises a polyglycol region and a lipid selected fromsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof the peptide is relaxin or a derivative thereof,the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof and thehalf-life of the modified therapeutic agent is longer than the half-lifeof the peptide alone. The cysteine residue may be an amino acid additionor substitution on the peptide. The mTA may be an LC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises an extended recombinant polypeptide (XTEN) comprising(i) an amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; and the half-life of the modified therapeuticagent is longer than the half-life of the peptide alone. The peptide maycomprise one or more amino acid additions, deletions, substitutions, ora combination thereof. The cysteine residue may be an amino acidaddition or substitution on the peptide. The cysteine residue may be anamino acid addition or substitution on a wild-type peptide. The mTA maybe an XTEN-modified therapeutic agent (XTEN-mTA).

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises an extended recombinant polypeptide (XTEN) comprising(i) an amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; the peptide is selected from relaxin, H1 relaxin,H2 relaxin, H3 relaxin, human INSL3, human INSL4, human INSL6, humanIGF1, human IGFII, human insulin, oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, leptin, betatrophin, FGF 21,GDF 11, ANGPTL3, peptide-based toxin, Moka, and VM-24, or a derivativethereof, the derivative being a peptide comprising one or more aminoacid additions, deletions, or substitutions, or a combination thereof;and the half-life of the modified therapeutic agent is longer than thehalf-life of the peptide alone. The cysteine residue may be an aminoacid addition or substitution on the peptide. The cysteine residue maybe an amino acid addition or substitution on a wild-type peptide. ThemTA may be an XTEN-modified therapeutic agent (XTEN-mTA).

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises an extended recombinant polypeptide (XTEN) comprising(i) an amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; the peptide is selected from relaxin,oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1 (GLP-1),GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, leptin, betatrophin, FGF 21, GDF 11, ANGPTL3, Toxin-550, Moka,and VM-24, or a derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; and the half-life of themodified therapeutic agent is longer than the half-life of the peptidealone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide. The mTA may be anXTEN-modified therapeutic agent (XTEN-mTA).

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises an extended recombinant polypeptide (XTEN) comprising(i) an amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; the peptide is relaxin or a derivative thereof,the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof; andthe half-life of the modified therapeutic agent is longer than thehalf-life of the peptide alone. The cysteine residue may be an aminoacid addition or substitution on the peptide. The cysteine residue maybe an amino acid addition or substitution on a wild-type peptide. ThemTA may be an XTEN-modified therapeutic agent (XTEN-mTA).

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent and one or more half-life extendingmoieties, wherein the therapeutic agent is a peptide that is covalentlyattached to each of the one or more half-life extending moieties via acysteine residue on the peptide; each of the half-life extendingmoieties comprises an extended recombinant polypeptide (XTEN) comprising(i) an amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; the therapeutic agent is encoded by an amino acidsequence comprising at least a portion of a polypeptide sequenceselected from a group consisting of SEQ ID NO: 10-56; and the half-lifeof the modified therapeutic agent is longer than the half-life of thepeptide alone. The cysteine residue may be an amino acid addition orsubstitution on the peptide. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide. The mTA may be anXTEN-modified therapeutic agent (XTEN-mTA).

The mTAs disclosed herein may have the structure:TA-A¹-P¹-X  Formula (II)wherein:

TA is the therapeutic agent;

A¹ is a chemical group linking TA and P¹ or X;

P¹ is a bond or comprises polyglycol; and

X is a half-life extending moiety.

X of Formula (II) may comprise a lipid.

X of formula (II) may comprise an extended recombinant polypeptide(XTEN).

The P¹ of an mTA of Formula (II) may be a bond.

A sulfur or nitrogen atom of an amino acid residue of TA may beconnected to A¹ via a chemical bond in an mTA of Formula (II). The A¹ ofan mTA of Formula (II) may be selected from

and

-   -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, —NC(O)R⁵,        —NC(O)OR⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl,        (cycloalkyl)alkyl, or heteroalkyl;    -   k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   q is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The P¹ of an mTA of Formula (II) may comprise polyglycol. The polyglycolmay be selected from polyethylene glycol, polypropylene glycol,polybutylene glycol, or a combination thereof. The polyglycol may bepolyethylene glycol. The polyglycol may be polypropylene glycol. Thepolyglycol may be polybutylene glycol.

The P¹ of an mTA of Formula (II) may be -PEG-A²-; wherein PEG is achemical group comprising one or more polyethyleneglycol subunits; andA² is a chemical group linking PEG and X. PEG may be selected from

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20. A² may be selected from a bond,

wherein X is a bond, NR⁵, S, or 0;

-   -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl, or        heteroalkyl; R⁶ is OH or —NR⁵R⁵;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

The half-life extending moiety of Formula (II) may comprise a lipid. Thelipid may be selected from a group consisting of propanoic acid,butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoicacid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid,tridecanoic acid, tetradecanoic acid, myristic acid, pentadecanoic acid,hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoicacid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoicacid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid,heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanoicacid, henatriacontanoic acid, dotriacontanoic acid, tritriacontanoicacid, tetratriacontanoic acid, pentatriacontanoic acid andhexatriacontanoic acid. The lipid may be selected from a groupconsisting of malonic acid, succinic acid, glutaric acid, adipic acid,pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioicacid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid,pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid,octadecanedioic acid, and nonadecanedioic acid. The lipid may beselected from a group consisting of myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentanoicacid, erucic acid, docosahexaenoic acid. The lipid may be selected froma group consisting of cholesterol, 7-OH cholesterol,7,25-dihydroxycholesterol, cholic acid, chenodeoxycholic acid,lithocholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholicacid, glycolithocholic acid, and glycochenodeoxycholic acid. The one ormore lipids of an mTA of Formula (II) may be selected from a groupconsisting of cholic acid, chenodeoxycholic acid, lithocholic acid,deoxycholic acid, glycocholic acid, glycodeoxycholic acid,glycolithocholic acid, and glycochenodeoxycholic acid. The one or morelipids of an mTA of Formula (II) may be selected from a group consistingof α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol,α-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol. The oneor more lipids of an mTA of Formula (II) may be selected from a groupconsisting of octadecanedioic acid, tetradecylamine, myristic acid,docosahexaenoic acid, lithocholic acid ester, cholic acid and palmiticacid.

The mTAs disclosed herein may have the structure:TA-A¹-P¹-X  Formula (IIa)wherein:

-   -   TA is the therapeutic agent with a cysteine residue, wherein the        cysteine residue is connected to A¹;    -   A¹ is a chemical group linking TA and P¹;    -   P¹ is a bond or -PEG-A²-;    -   PEG is a chemical group comprising one or more        polyethyleneglycol subunits;    -   A² is a chemical group linking PEG and X; and    -   X is a half-life extending moiety.

X of Formula IIa) may comprise a lipid.

X of formula IIa) may comprise an extended recombinant polypeptide(XTEN).

The sulfur atom of the cysteine residue of the TA of an mTA of FormulaIIa) may be connected to A¹ via a chemical bond.

The PEG of an mTA of Formula IIa) may be selected from:

wherein

-   m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,    14, 15, 16, 17, 18, 19, or 20.

In some embodiments of an mTA of Formula IIa) disclosed herein,

-   -   A¹ is selected from and

-   -   R¹, R², R³, and R⁴ are independently selected from H, halo, CN,        —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;    -   p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   q is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments described of an mTA of Formula IIa) disclosedherein,

-   -   A² is selected from a bond,

-   -   X is a bond, NR⁵, or 0;    -   R¹, R², R³, and R⁴ are independently selected from H, halo, CN,        —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

In some embodiments of an mTA of Formula IIa) disclosed herein, P¹ is-PEG-A².

Disclosed herein are methods of producing an mTA of Formula (II), themethod comprising reacting the cysteine residue of TA with A³-P¹-X,wherein A³ is a reactive precursor to form A¹. In some embodiments, A³is a haloacetamide, maleimide, benzyl halide, or pyridyl disulfide. Infurther embodiments, A³ is a haloacetamide. In still furtherembodiments, A³ is a bromoacetamide.

The modified therapeutic agents (mTAs) may comprise (a) one or morelipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols; and (b) one or more therapeutic agents (TAs),wherein the one or more lipids are conjugated to the one or moretherapeutic agents via a cysteine residue.

The modified therapeutic agents (mTAs) may comprise (a) one or morelipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,fatty amines, and fatty alcohols, and derivatives thereof, and (b) atherapeutic agent (TA), wherein the TA is a peptide and the one or morelipids are conjugated to the TA via an amino acid residue on thepeptide. The amino acid residue may be a cysteine, lysine, or serine.The amino acid residue may be selected from cysteine or lysine. Theamino acid residue may be cysteine. The amino acid residue may belysine. The amino acid may be an amino acid mutation. The amino acid maybe an amino acid addition or an amino acid substitution.

The modified therapeutic agents (mTA) may comprise (a) one or morelipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols; and (b) one or more therapeutic agents (TAs),wherein the one or more TAs comprise a relaxin peptide or derivativethereof. The modified therapeutic agents (mTA) may comprise (a) one ormore lipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,fatty amines, and fatty alcohols, and derivatives thereof; and (b) atherapeutic agent (TA), wherein the TA comprises a relaxin peptide orderivative thereof.

The modified therapeutic agents (mTA) may comprise (a) one or morelipids, the lipids selected from sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols; and (b) one or more therapeutic agents (TAs),wherein the one or more TAs comprise a relaxin peptide or derivativethereof, wherein the one or more lipids are attached to the one or moretherapeutic agent via a cysteine residue. The modified therapeuticagents (mTA) may comprise (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, fatty amines, and fattyalcohols, and derivatives thereof, and (b) a therapeutic agent (TA),wherein the TA comprises a relaxin peptide or derivative thereof,wherein the one or more lipids are attached to the therapeutic agent viaan amino acid residue on the peptide. The amino acid residue may be acysteine, lysine, or serine. The amino acid residue may be selected fromcysteine or lysine. The amino acid residue may be cysteine. The aminoacid residue may be lysine. The amino acid may be an amino acidmutation. The amino acid may be an amino acid addition or an amino acidsubstitution. The amino acid residue may be an amino acid addition orsubstitution on a wild-type peptide.

The modified therapeutic agents (mTA) may comprise one or more lipidsattached to one or more therapeutic agents (TAs), wherein the one ormore TAs comprise a modified relaxin peptide, and wherein the modifiedrelaxin peptide comprises a wild-type relaxin polypeptide with one ormore amino acid mutations. The modified therapeutic agents (mTA) maycomprise one or more lipids attached to a therapeutic agent (TA),wherein the TA comprises a modified relaxin peptide, and wherein themodified relaxin peptide comprises a wild-type relaxin polypeptide withone or more amino acid mutations.

The mTAs disclosed herein may comprise a TA comprising a modifiedrelaxin peptide. The mTAs disclosed herein may comprise one or morelipids, wherein the one or more lipids may be selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty acids and fatty alcohols, and derivatives thereof.The attachment of the one or more lipids to the modified relaxin peptidemay comprise covalent attachment. The one or more lipids may be attachedto the modified relaxin peptide via a cysteine residue. The cysteineresidue may be an amino acid mutation. The cysteine residue may be anamino acid addition or an amino acid substitution. The cysteine residuemay be an amino acid addition or substitution on a wild-type peptide.

Half-Life Extending Moieties

Disclosed herein are modified therapeutic agents (mTAs) comprising atherapeutic agent and one or more half-life extending moieties, whereinthe therapeutic agent is a peptide that is covalently attached to eachof the one or more half-life extending moieties via a cysteine residueon the peptide; and the half-life of the modified therapeutic agent islonger than the half-life of the peptide alone.

The one or more half-life extending moieties may comprise a lipid, apolyglycol region, or a combination thereof. The one or more half-lifeextending moieties may comprise an extended recombinant polypeptide(XTEN).

The polyglycol region may comprise one or more polyethylene glycolunits, polypropylene glycol units, or polybutylene glycol units, or acombination thereof. The polyglycol region may comprise one or morepolyethylene glycol units. The polyglycol region may comprise one ormore polypropylene glycol units. The polyglycol region may comprise oneor more polybutylene glycol units.

The polyglycol region may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or more polyethylene glycol units, polypropylene glycol units, orpolybutylene glycol units, or a combination thereof. The polyglycolregion may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or morepolyethylene glycol units. The polyglycol region may comprise 10, 20,30, 40, 50, 60, 70, 80, 90, 100, or more polypropylene glycol units. Thepolyglycol region may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,or more polybutylene glycol units.

The polyglycol region may comprise 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. Thepolyglycol region may comprise 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, or more polyethylene glycol units. The polyglycol region maycomprise 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or morepolypropylene glycol units. The polyglycol region may comprise 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, or more polybutylene glycolunits.

The polyglycol region may comprise 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000,17000, 18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, ormore polyethylene glycol units, polypropylene glycol units, orpolybutylene glycol units, or a combination thereof. The polyglycolregion may comprise 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000,19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, or morepolyethylene glycol units. The polyglycol region may comprise 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000,13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000,35000, 40000, 45000, 50000, or more polypropylene glycol units. Thepolyglycol region may comprise 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000,18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, or morepolybutylene glycol units.

The polyglycol region may comprise a molecular weight of 500-50,000daltons. The polyglycol region may comprise a molecular weight of500-40,000 daltons. The polyglycol region may comprise a molecularweight of 500-30,000 daltons. The polyglycol region may comprise amolecular weight of 500-20,000 daltons. The polyglycol region maycomprise a molecular weight of 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000,30000, 35000, 40000, or 45000 daltons or more, including incrementstherein.

The half-life extending moieties may be attached to the TA via acysteine residue on the TA. The attachment may be via chemicalattachment through the sulfur atom of a cysteine residue on the TA. Thehalf-life extending moieties may comprise a linker which is directlyattached to the sulfur atom of a cysteine residue on the TA. Thehalf-life extending moieties may comprise a linker which is covalentlyattached to the sulfur atom of a cysteine residue on the TA.

Lipid Conjugate (LC)

Disclosed herein are lipid conjugates (LCs) comprising one or morelipids attached to one or more therapeutic agents (TAs). The lipidconjugate may comprise one or more lipids attached to one TA. The lipidconjugate may further comprise a hydrophilic connector between the oneor more TAs and the one or more lipids. The lipid conjugate may furthercomprise one or more polyethyleneglycol subunits. The one or more lipidsmay be pegylated.

The LCs disclosed herein may have the structure:TA-A¹P¹L  Formula (I)wherein:

TA is the therapeutic agent;

A¹ is a chemical group linking TA and P¹ or L;

P¹ is a bond or comprises polyglycol; and

L is the lipid.

The P¹ of an LC of Formula (I) may be a bond.

A sulfur or nitrogen atom of an amino acid residue of TA may beconnected to A¹ via a chemical bond in an LC of Formula (I). The A¹ ofan LC of Formula (I) may be selected from

and

-   -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, —NC(O)R⁵,        —NC(O)OR⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl,        (cycloalkyl)alkyl, or heteroalkyl;    -   k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   q is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The P¹ of an LC of Formula (I) may comprise polyglycol. The polyglycolmay be selected from polyethylene glycol, polypropylene glycol,polybutylene glycol, or a combination thereof. The polyglycol may bepolyethylene glycol. The polyglycol may be polypropylene glycol. Thepolyglycol may be polybutylene glycol.

The P¹ of an LC of Formula (I) may be -PEG-A²-; wherein PEG is achemical group comprising one or more polyethyleneglycol subunits; andA² is a chemical group linking PEG

and L. PEG may be selected from wherein m and n are independently 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. A²may be selected from a bond,

wherein X is a bond, NR⁵, S, or 0;

-   -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl, or        heteroalkyl; R⁶ is OH or —NR⁵R⁵;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

The one or more lipids of an LC of Formula (I) may be selected from agroup consisting of propanoic acid, butanoic acid, pentanoic acid,hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoicacid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoicacid, myristic acid, pentadecanoic acid, hexadecanoic acid,heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoicacid, heneicosanoic acid, docosanoic acid, tricosanoic acid,tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoicacid, octacosanoic acid, nonacosanoic acid, triacontanoic acid,henatriacontanoic acid, dotriacontanoic acid, tritriacontanoic acid,tetratriacontanoic acid, pentatriacontanoic acid and hexatriacontanoicacid. The one or more lipids of an LC of Formula (I) may be selectedfrom a group consisting of malonic acid, succinic acid, glutaric acid,adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid,undecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid,heptadecanedioic acid, octadecanedioic acid, and nonadecanedioic acid.The one or more lipids of an LC of Formula (I) may be selected from agroup consisting of myristoleic acid, palmitoleic acid, sapienic acid,oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidicacid, α-linolenic acid, arachidonic acid, eicosapentanoic acid, erucicacid, docosahexaenoic acid. The one or more lipids of an LC of Formula(I) may be selected from a group consisting of cholesterol, 7-OHcholesterol, 7,25-dihydroxycholesterol, cholic acid, chenodeoxycholicacid, lithocholic acid, deoxycholic acid, glycocholic acid,glycodeoxycholic acid, glycolithocholic acid, and glycochenodeoxycholicacid. The one or more lipids of an LC of Formula (I) may be selectedfrom a group consisting of cholic acid, chenodeoxycholic acid,lithocholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholicacid, glycolithocholic acid, and glycochenodeoxycholic acid. The one ormore lipids of an LC of Formula (I) may be selected from a groupconsisting of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol,α-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol. The oneor more lipids of an LC of Formula (I) may be selected from a groupconsisting of octadecanedioic acid, tetradecylamine, myristic acid,docosahexaenoic acid, lithocholic acid ester, cholic acid and palmiticacid.

The LCs disclosed herein may have the structure:TA-A¹P¹L  Formula (Ia)wherein:

-   -   TA is the therapeutic agent with a cysteine residue, wherein the        cysteine residue is connected to A¹;    -   A¹ is a chemical group linking TA and P¹;    -   P¹ is a bond or -PEG-A²-;    -   PEG is a chemical group comprising one or more        polyethyleneglycol subunits;    -   A² is a chemical group linking PEG and L; and    -   L is the lipid.

The sulfur atom of the cysteine residue of the TA of an LC of Formula(Ia) may be connected to A¹ via a chemical bond.

The PEG of an LC of Formula (Ia) may be selected from:

wherein

-   -   m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, or 20.

In some embodiments of an LC of Formula (Ia) disclosed herein,

A¹ is selected from

and

-   -   R¹, R², R³, and R⁴ are independently selected from H, halo, CN,        —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;    -   p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   q is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments described of an LC of Formula (Ia) disclosed herein,

A² is selected from a bond,

X is a bond, NR⁵, or 0;

-   -   R¹, R², R³, and R⁴ are independently selected from H, halo, CN,        —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

In some embodiments of an LC of Formula (Ia) disclosed herein, P¹ is-PEG-A².

Disclosed herein are methods of producing an LC of Formula (I), themethod comprising reacting the cysteine residue of TA with A³-P¹-L,wherein A³ is a reactive precursor to form A¹. In some embodiments, A³is a haloacetamide, maleimide, benzyl halide, or pyridyl disulfide. Infurther embodiments, A³ is a haloacetamide. In still furtherembodiments, A³ is a bromoacetamide.

The lipid conjugates (LCs) may comprise (a) one or more lipids, thelipids selected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more lipids are conjugated to the one or more therapeutic agents viaa cysteine residue.

The lipid conjugates (LCs) may comprise (a) one or more lipids, thelipids selected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof; and (b) a therapeutic agent(TA), wherein the TA is a peptide and the one or more lipids areconjugated to the TA via an amino acid residue on the peptide. The aminoacid residue may be a cysteine, lysine, or serine. The amino acidresidue may be selected from cysteine or lysine. The amino acid residuemay be cysteine. The amino acid residue may be lysine. The amino acidmay be an amino acid mutation. The amino acid may be an amino acidaddition or an amino acid substitution.

The lipid conjugates (LC) may comprise (a) one or more lipids, thelipids selected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more TAs comprise a relaxin peptide or derivative thereof. The lipidconjugates (LC) may comprise (a) one or more lipids, the lipids selectedfrom sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, fatty amines, and fattyalcohols, and derivatives thereof and (b) a therapeutic agent (TA),wherein the TA comprises a relaxin peptide or derivative thereof.

The lipid conjugates (LC) may comprise (a) one or more lipids, thelipids selected from sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more TAs comprise a relaxin peptide or derivative thereof, whereinthe one or more lipids are attached to the one or more therapeutic agentvia a cysteine residue. The lipid conjugates (LC) may comprise (a) oneor more lipids, the lipids selected from sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, fatty amines, and fatty alcohols, and derivatives thereof and(b) a therapeutic agent (TA), wherein the TA comprises a relaxin peptideor derivative thereof, wherein the one or more lipids are attached tothe therapeutic agent via an amino acid residue on the peptide. Theamino acid residue may be a cysteine, lysine, or serine. The amino acidresidue may be selected from cysteine or lysine. The amino acid residuemay be cysteine. The amino acid residue may be lysine. The amino acidmay be an amino acid mutation. The amino acid may be an amino acidaddition or an amino acid substitution.

The lipid conjugates (LC) may comprise one or more lipids attached toone or more therapeutic agents (TAs), wherein the one or more TAscomprise a modified relaxin peptide, and wherein the modified relaxinpeptide comprises a wild-type relaxin polypeptide with one or more aminoacid mutations. The lipid conjugates (LC) may comprise one or morelipids attached to a therapeutic agent (TA), wherein the TA comprises amodified relaxin peptide, and wherein the modified relaxin peptidecomprises a wild-type relaxin polypeptide with one or more amino acidmutations.

The LCs disclosed herein may comprise a TA comprising a modified relaxinpeptide. The LCs disclosed herein may comprise one or more lipids,wherein the one or more lipids may be selected from a group consistingof sterols, sterol derivatives, bile acids, vitamin E derivatives, fattyacids and fatty alcohols, and derivatives thereof. The attachment of theone or more lipids to the modified relaxin peptide may comprise covalentattachment. The one or more lipids may be attached to the modifiedrelaxin peptide via a cysteine residue. The cysteine residue may be anamino acid mutation. The cysteine residue may be an amino acid additionor an amino acid substitution. The cysteine residue may be an amino acidaddition or substitution on a wild-type peptide.

Linker

The LCs disclosed herein may further comprise one or more linkers. TheLCs disclosed herein may further comprise two or more linkers. The LCsdisclosed herein may further comprise three or more linkers. The LCsdisclosed herein may further comprise four or more linkers. LCsdisclosed herein may further comprise five or more linkers.

The one or more linkers may enable attachment of a lipid to atherapeutic agent. The linker may enable attachment of a lipid toanother lipid. The linker may enable attachment of a lipid to a chemicalgroup comprising one or more polyethyleneglycol subunits. The linker mayenable attachment of a PEG to another PEG. The linker may enableattachment of a PEG to a therapeutic agent. The linker may enableattachment of a therapeutic agent to another therapeutic agent. Thelinker may be an amino acid. The linker may be an amino acid of thetherapeutic agent. The linker may be an amino acid mutation of thetherapeutic agent. The linker may be a substituted amino acid of thetherapeutic agent. The linker may be an amino acid addition of thetherapeutic agent. The linker may be an amino acid mutation located atthe C-terminus of the peptide. The linker may be an amino acid mutationlocated at the N-terminus of the peptide. The linker may be an aminoacid mutation located at a non-terminus position of the peptide. Thelinker may be a lysine. The linker may be a cysteine. The linker may bean L-cysteine. The linker may be an ether or an amide. The linker maylink a PEG molecule to a lipid.

Lipid Derivatives

The LCs disclosed herein may comprise one or more lipid derivatives. Thelipid derivatives may be attached to a TA. Attachment of the lipidderivative to the TAs may enhance the pharmacokinetic properties of theTAs. Lipid derivatives may comprise polyglycol. Lipid derivatives may bepegylated. A pegylated lipid may comprise at least onepolyethyleneglycol subunit. The lipid derivatives may be not pegylated.Lipids may be broadly defined as hydrophobic or amphiphilic smallmolecules. Lipids may be naturally occurring or synthetic. Lipids may beeicosanoids, prostaglandins, leukotrienes, thromboxanes, wax esters,coenzyme A derivatives, fatty acid carnitines, fatty acid amides,ethanolamines, bile acids, vitamin E, vitamin A, vitamin D, vitamin K,fat-soluble vitamin derivatives, monoglycerides, diglycerides,triglycerides, phospholipids, phosphatidylcholine, glycerolipids,glycerols, glycerophospholypids, sphingolipids, saccharolipids,polyketides, sterols, sterol derivatives, sterol lipids, steroidhormones, prenol lipids, carotenoids, fatty acids, and fatty alcohols.

In one aspect, disclosed herein are lipid derivatives having thestructure of A³-P¹-L, wherein:

-   -   A³ is a haloacetamide, maleimide, benzyl halide, or pyridyl        disulfide;    -   P¹ is a bond or -PEG-A²-;    -   PEG is a chemical group comprising one or more        polyethyleneglycol subunits;    -   A² is a chemical group linking PEG and L; and    -   L is a lipid selected from sterols, sterol derivatives, bile        acids, vitamin E derivatives, fatty di-acids, fatty acids, fatty        amides, and fatty alcohols, and derivatives thereof.

In some embodiments described herein, PEG is selected from:

wherein

-   -   m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, or 20.

In some embodiments described herein,

-   -   A² is selected from a bond,

-   -   X is a bond, NR⁵, S, or 0;    -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl, or        heteroalkyl; R⁶ is OH or —NR⁵R⁵;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

Lipids

The LCs disclosed herein may comprise one or more lipids. The one ormore lipids may be fatty acids. The fatty acids (FAs) may be attached toone or more therapeutic agents (TAs). Attachment of the fatty acids tothe TAs may enhance the pharmacokinetic properties of the TAs. Fattyacids may be fatty di-acids, fatty amides, fatty amines, or fattyalcohols. Fatty acids may be saturated or unsaturated. Saturated fattyacids include, but are not limited to, lauric acid, myristic acid,palmitic acid, stearic acid, arachidic acid. Unsaturated fatty acidsinclude, but are not limited to palmitoleic acid, oleic acid, linoleicacid, linolenic acid, erucic acid and arachidonic acid. Fatty acids maybe short-chain fatty acids, medium chain fatty acids, long chain fattyacids or very long chain fatty acids. Fatty acids may be monounsaturatedor polyunsaturated. Fatty acids may be omega fatty acids, essentialfatty acids, partially hydrogenated fatty acids, cis-isomer fatty acids,or trans-isomer fatty acids. Fatty acids may be omega-3 fatty acids,omega-6 fatty acids or omega-9 fatty acids. Fatty acids may bedicarboxylic acids.

The fatty acid may comprise a chain of about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more carbon atoms.The fatty acid may comprise a carbon chain further comprising 1, 2, 3,4, 5, 6 or more double bonds. The fatty acid may be naturally occurring.The fatty acid may not be naturally occurring. The fatty acid may besynthesized.

The LCs disclosed herein may further comprise one or more fatty acids.The LCs disclosed herein may further comprise two or more fatty acids.The LCs disclosed herein may further comprise three or more fatty acids.The LCs disclosed herein may further comprise four or more fatty acids.LCs disclosed herein may further comprise five or more fatty acids. Thefatty acids may be different. The fatty acids may be the same.

The one or more lipids of any LC may be selected from the groupconsisting of myristic acid, docosahexanoic acid, lithocholic acidester, cholic acid and palmitic acid. The one or more lipids of any LCmay be myristic acid. The one or more lipids of any LC may bedocosahexanoic acid. The one or more lipids of any LC may be lithocholicacid ester. The one or more lipids of any LC may be cholic acid. The oneor more lipids of any LC may be palmitic acid.

The LCs may comprise one or more sterols or sterol derivatives. Thesterols or sterol derivatives may be selected from a group consisting ofcholesterol, 7-OH cholesterol, 7,25-dihydroxycholesterol, cholic acid,chenodeoxycholic acid, lithocholic acid, deoxycholic acid, glycocholicacid, glycodeoxycholic acid, glycolithocholic acid, andglycochenodeoxycholic acid.

The LCs may comprise one or more bile acids. The bile acids may beselected from a group consisting of cholic acid, chenodeoxycholic acid,lithocholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholicacid, glycolithocholic acid, and glycochenodeoxycholic acid.

The LC may comprise one or more Vitamin E derivatives. The Vitamin Ederivatives may be selected from a group consisting of α-tocopherol,β-tocopherol, γ-tocopherol, tocopherol, α-tocotrienol, β-tocotrienol,γ-tocotrienol and δ-tocotrienol.

Pegylated Lipid

The LCs disclosed herein in may comprise one or more pegylated lipids. Apegylated lipid may comprise at least one polyethyleneglycol subunit.The connection between the lipid and the one or more polyethyleneglycolsubunits may be a direct bond or a linker (A²). Non-limiting examples ofa linker between the lipid and the one or more polyethyleneglycolsubunits include: a bond,

wherein

-   -   X is a bond, NR⁵, S, or 0;    -   each R¹, R², R³, and R⁴ is independently selected from H, halo,        CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;    -   each R⁵ is independently H, alkyl, haloalkyl, arylalkyl, or        heteroalkyl;    -   R⁶ is OH or —NR⁵R⁵;    -   each R⁷ is independently selected from H, alkyl, haloalkyl,        arylalkyl, and heteroalkyl;    -   r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   s is 1, 2, 3, 4, or 5.

A pegylated lipid may have the structure P¹-L, wherein P¹ is -PEG-A²-;PEG is a chemical group comprising one or more polyethyleneglycolsubunits; A² is a chemical group linking PEG and L; and L is a lipid.PEG may be selected from:

wherein

m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

A pegylated lipid may be connected to a therapeutic agent through alinker. The linker may comprise one or more amide moieties.

XTEN-Modified Therapeutic Agent (XTEN-mTA)

Disclosed herein is an XTEN-modified therapeutic agent (XTEN-mTA)comprising (a) an extended recombinant polypeptide (XTEN); and (b) atherapeutic agent (TA) selected from a group consisting of relaxin,fibroblast growth factor 21 (FGF21), leptin, Toxin-550, and analogsthereof.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) an extended recombinant polypeptide (XTEN);and (b) a therapeutic agent (TA) comprising relaxin or analog thereof.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) an extended recombinant polypeptide (XTEN);and (b) a therapeutic agent (TA) comprising FGF21 or analog thereof.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) an extended recombinant polypeptide (XTEN);and (b) a therapeutic agent (TA) comprising leptin or analog thereof.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) an extended recombinant polypeptide (XTEN);and (b) a therapeutic agent (TA) comprising Toxin-550 or analog thereof.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) a therapeutic agent (TA) selected from a groupconsisting of relaxin, fibroblast growth factor 21 (FGF21), leptin,Toxin-550, and analogs thereof; and (b) an extended recombinantpolypeptide (XTEN) comprising (i) an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 70% of the total amino acid sequence; (ii) asubstantially non-repetitive amino acid sequence; (iii) an amino acidsequence that has less than about 10% alpha helices; and/or (iv) anamino acid sequence that has less than about 10% beta-sheets, whereinthe TA is attached to the XTEN. Attachment of the TA to the XTEN may bevia a cysteine residue on the TA. Attachment of the TA to the XTEN maybe through the sulfur atom of a cysteine residue on the TA.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) a therapeutic agent (TA) comprising relaxin oran analog thereof; and an extended recombinant polypeptide (XTEN)comprising (i) an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 70% of the total amino acid sequence; (ii) a substantiallynon-repetitive amino acid sequence; (iii) an amino acid sequence thathas less than about 10% alpha helices; and/or (iv) an amino acidsequence that has less than about 10% beta-sheets, wherein the TA isattached to the XTEN. Attachment of the TA to the XTEN may be via acysteine residue on the TA. Attachment of the TA to the XTEN may bethrough the sulfur atom of a cysteine residue on the TA.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) a therapeutic agent (TA) comprising FGF21 oran analog thereof; and an extended recombinant polypeptide (XTEN)comprising (i) an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 70% of the total amino acid sequence; (ii) a substantiallynon-repetitive amino acid sequence; (iii) an amino acid sequence thathas less than about 10% alpha helices; and/or (iv) an amino acidsequence that has less than about 10% beta-sheets, wherein the TA isattached to the XTEN. Attachment of the TA to the XTEN may be via acysteine residue on the TA. Attachment of the TA to the XTEN may bethrough the sulfur atom of a cysteine residue on the TA.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) a therapeutic agent (TA) comprising leptin oran analog thereof; and an extended recombinant polypeptide (XTEN)comprising (i) an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 70% of the total amino acid sequence; (ii) a substantiallynon-repetitive amino acid sequence; (iii) an amino acid sequence thathas less than about 10% alpha helices; and/or (iv) an amino acidsequence that has less than about 10% beta-sheets, wherein the TA isattached to the XTEN. Attachment of the TA to the XTEN may be via acysteine residue on the TA. Attachment of the TA to the XTEN may bethrough the sulfur atom of a cysteine residue on the TA.

Further disclosed herein is an XTEN-modified therapeutic agent(XTEN-mTA) comprising (a) a therapeutic agent (TA) comprising Toxin-550or an analog thereof; and an extended recombinant polypeptide (XTEN)comprising (i) an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 70% of the total amino acid sequence; (ii) a substantiallynon-repetitive amino acid sequence; (iii) an amino acid sequence thathas less than about 10% alpha helices; and/or (iv) an amino acidsequence that has less than about 10% beta-sheets, wherein the TA isattached to the XTEN. Attachment of the TA to the XTEN may be via acysteine residue on the TA. Attachment of the TA to the XTEN may bethrough the sulfur atom of a cysteine residue on the TA.

The XTEN-mTA may comprise less than about 2000, 1800, 1600, 1500, 1400,1300, 1200, 1100, 1000 amino acids in length. The XTEN-mTA may compriseless than about 975, 950, 925, 875, 850, 825, 800, 775, 750, 725, 700,675, 650, 625, or 600 amino acids in length. The XTEN-mTA may compriseless than about 900 amino acids in length. The XTEN-mTA may compriseless than about 890 amino acids in length. The XTEN-mTA may compriseless than about 880 amino acids in length. The XTEN-mTA may compriseless than about 870 amino acids in length. The XTEN-mTA may compriseless than about 860 amino acids in length. The XTEN-mTA may compriseless than about 850 amino acids in length.

The amino sequence of the XTEN-mTA may comprise one or more aspartateresidues. The amino sequence of the XTEN-mTA may comprise 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more aspartateresidues. The amino sequence of the XTEN-mTA may comprise 2 or moreaspartate residues. The amino sequence of the XTEN-mTA may comprise 3 ormore aspartate residues. The amino sequence of the XTEN-mTA may comprise4 or more aspartate residues.

The XTEN-mTA may comprise one or more XTENs. The XTEN-mTA may comprise2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore XTENs. The XTEN-mTA may comprise 2 or more XTENs. The XTEN-mTA maycomprise 3 or more XTENs. The XTEN-mTA may comprise 4 or more XTENs.

The XTEN may comprise an amino acid sequence comprising 10 or more aminoacids based on or derived from the amino acid sequence selected from agroup consisting of SEQ ID NOs: 66-67. The XTEN may comprise an aminoacid sequence comprising 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or more amino acidsbased on or derived from the amino acid sequence selected from a groupconsisting of SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence comprising 20 or more amino acids based on or derived from theamino acid sequence selected from a group consisting of SEQ ID NOs:66-67. The XTEN may comprise an amino acid sequence comprising 30 ormore amino acids based on or derived from the amino acid sequenceselected from a group consisting of SEQ ID NOs: 66-67. The XTEN maycomprise an amino acid sequence comprising 40 or more amino acids basedon or derived from the amino acid sequence selected from a groupconsisting of SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence comprising 50 or more amino acids based on or derived from theamino acid sequence selected from a group consisting of SEQ ID NOs:66-67. The XTEN may comprise an amino acid sequence comprising 60 ormore amino acids based on or derived from the amino acid sequenceselected from a group consisting of SEQ ID NOs: 66-67.

The XTEN may comprise an amino acid sequence less than about 400 aminoacids. The XTEN may comprise an amino acid sequence less than about 390,380, 375, 370, 365, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270,260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130,120, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids. The XTENmay comprise an amino acid sequence less than about 350 amino acids. TheXTEN may comprise an amino acid sequence less than about 300 aminoacids. The XTEN may comprise an amino acid sequence less than about 250amino acids.

The XTEN may comprise an amino acid sequence that may be at least about50% homologous to an amino acid sequence selected from a groupconsisting of SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that may be at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, or 99% homologous to an amino acid sequence selectedfrom a group consisting of SEQ ID NOs: 66-67. The XTEN may comprise anamino acid sequence that may be at least about 60% homologous to anamino acid sequence selected from a group consisting of SEQ ID NOs:66-67. The XTEN may comprise an amino acid sequence that may be at leastabout 70% homologous to an amino acid sequence selected from a groupconsisting of SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that may be at least about 80% homologous to an amino acidsequence selected from a group consisting of SEQ ID NOs: 66-67.

The XTEN may comprise an amino acid sequence characterized in that thesum of glycine (G), alanine (A), serine (S), threonine (T), glutamate(E), aspartate (D), leucine (L) and proline (P) residues constitutesmore than about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99%the total amino acid sequence. The XTEN may comprise an amino acidsequence characterized in that the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E), aspartate (D), leucine (L) andproline (P) residues constitutes more than about 75% of the total aminoacid sequence. The XTEN may comprise an amino acid sequencecharacterized in that the sum of glycine (G), alanine (A), serine (S),threonine (T), glutamate (E), aspartate (D), leucine (L) and proline (P)residues constitutes more than about 80% of the total amino acidsequence. The XTEN may comprise an amino acid sequence characterized inthat the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 85% of the total amino acid sequence. TheXTEN may comprise an amino acid sequence characterized in that the sumof glycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 90% of the total amino acid sequence. The XTEN may comprisean amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 95% ofthe total amino acid sequence.

The XTEN may be attached to the N-terminus, C-terminus, or the N- andC-terminus of the TA. The XTEN may be attached to a cysteine residuelocated at the N- or C-terminus of the TA. The XTEN may be chemicallyattached to a cysteine residue located at the N- or C-terminus of theTA. The XTEN may be attached to the sulfur atom of a cysteine residuelocated at the N- or C-terminus of the TA. The XTEN may be chemicallyattached to the sulfur atom of a cysteine residue located at the N- orC-terminus of the TA. The XTEN may be attached to an internal residue ofthe TA. The XTEN may be attached to a cysteine residue located at aninternal position of the TA. The XTEN may be chemically attached to acysteine residue located at an internal position of the TA. The XTEN maybe attached to the sulfur atom of a cysteine residue located at aninternal position of the TA. The XTEN may be chemically attached to thesulfur atom of a cysteine residue located at internal position of theTA.

The XTEN-mTAs may comprise one or more TAs. The XTEN-mTA may comprise 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moreTAs. The XTEN-mTA may comprise 2 or more TAs. The XTEN-mTA may comprise3 or more TAs. The XTEN-mTA may comprise 4 or more TAs.

The TA may comprise an amino acid sequence comprising at least a portionof a polypetide sequence selected from a group consisting of SEQ ID NOs:10-56. The TA may comprise an amino acid sequence comprising 10 or moreamino acids based on or derived from the amino acid sequence selectedfrom a group consisting of SEQ ID NOs: 10-56. The TA may comprise anamino acid sequence comprising 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180 or more amino acids based on orderived from the amino acid sequence selected from a group consisting ofSEQ ID NOs: 10-56. The TA may comprise an amino acid sequence comprising20 or more amino acids based on or derived from the amino acid sequenceselected from a group consisting of SEQ ID NOs: 10-56. The TA maycomprise an amino acid sequence comprising 30 or more amino acids basedon or derived from the amino acid sequence selected from a groupconsisting of SEQ ID NOs: 10-56. The TA may comprise an amino acidsequence comprising 50 or more amino acids based on or derived from theamino acid sequence selected from a group consisting of SEQ ID NOs:10-56. The TA may comprise an amino acid sequence comprising 70 or moreamino acids based on or derived from the amino acid sequence selectedfrom a group consisting of SEQ ID NOs: 10-56. The amino acids based onor derived from the amino acid sequence selected from a group consistingof SEQ ID NOs: 10-56 may be consecutive. amino acids based on or derivedfrom the amino acid sequence selected from a group consisting of SEQ IDNOs: 10-56 may be non-consecutive.

The TA may comprise an amino acid sequence less than about 400 aminoacids. The TA may comprise an amino acid sequence less than about 375,350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130,120, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids.

The TA may comprise an amino acid sequence that is at least about 50%homologous to an amino acid sequence selected from a group consisting ofSEQ ID NOs: 10-56. The TA may comprise an amino acid sequence that is atleast about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, or99% homologous to an amino acid sequence selected from a groupconsisting of SEQ ID NOs: 10-56. The TA may comprise an amino acidsequence that is at least about 60% homologous to an amino acid sequenceselected from a group consisting of SEQ ID NOs: 10-56. The TA maycomprise an amino acid sequence that is at least about 70% homologous toan amino acid sequence selected from a group consisting of SEQ ID NOs:10-56. The TA may comprise an amino acid sequence that is at least about80% homologous to an amino acid sequence selected from a groupconsisting of SEQ ID NOs: 10-56. The TA may comprise an amino acidsequence that is at least about 90% homologous to an amino acid sequenceselected from a group consisting of SEQ ID NOs: 10-56.

The TA may be relaxin or an analog thereof. The XTEN may be attached tothe N-terminus, C-terminus, or the N- and C-terminus of relaxin or ananalog thereof. The XTEN may be attached to a cysteine residue locatedat the N- or C-terminus of relaxin or an analog thereof. The XTEN may bechemically attached to a cysteine residue located at the N- orC-terminus of relaxin or an analog thereof. The XTEN may be attached tothe sulfur atom of a cysteine residue located at the N- or C-terminus ofrelaxin or an analog thereof. The XTEN may be chemically attached to thesulfur atom of a cysteine residue located at the N- or C-terminus ofrelaxin or an analog thereof. The XTEN may be attached to a cysteineresidue located at the N- or C-terminus of relaxin or an analog thereof.The XTEN may be chemically attached to a cysteine residue located at aninternal position of relaxin or an analog thereof. The XTEN may beattached to the sulfur atom of a cysteine residue located at an internalposition of relaxin or an analog thereof. The XTEN may be chemicallyattached to the sulfur atom of a cysteine residue located at an internalposition of relaxin or an analog thereof.

The TA may be FGF21 or an analog thereof. The XTEN may be attached tothe N-terminus, C-terminus, or the N- and C-terminus of FGF21 or ananalog thereof. The XTEN may be attached to a cysteine residue locatedat the N- or C-terminus of FGF21 or an analog thereof. The XTEN may bechemically attached to a cysteine residue located at the N- orC-terminus of FGF21 or an analog thereof. The XTEN may be attached tothe sulfur atom of a cysteine residue located at the N- or C-terminus ofFGF21 or an analog thereof. The XTEN may be chemically attached to thesulfur atom of a cysteine residue located at the N- or C-terminus ofFGF21 or an analog thereof. The XTEN may be attached to an internalresidue of FGF1 or an analog thereof. The XTEN may be attached to acysteine residue located at an internal position of FGF21 or an analogthereof. The XTEN may be chemically attached to a cysteine residuelocated at an internal position of FGF21 or an analog thereof. The XTENmay be attached to the sulfur atom of a cysteine residue located at aninternal position of FGF21 or an analog thereof. The XTEN may bechemically attached to the sulfur atom of a cysteine residue located atan internal position of FGF21 or an analog thereof.

The TA may be leptin or an analog thereof. The XTEN may be attached tothe N-terminus, C-terminus, or the N- and C-terminus of leptin or ananalog thereof. The XTEN may be attached to a cysteine residue locatedat the N- or C-terminus of leptin or an analog thereof. The XTEN may bechemically attached to a cysteine residue located at the N- orC-terminus of leptin or an analog thereof. The XTEN may be attached tothe sulfur atom of a cysteine residue located at the N- or C-terminus ofleptin or an analog thereof. The XTEN may be chemically attached to thesulfur atom of a cysteine residue located at the N- or C-terminus ofleptin or an analog thereof. The XTEN may be attached to an internalresidue of leptin or an analog thereof. The XTEN may be attached to acysteine residue located at an internal position of leptin or an analogthereof. The XTEN may be chemically attached to a cysteine residuelocated at an internal position of leptin or an analog thereof. The XTENmay be attached to the sulfur atom of a cysteine residue located at aninternal position of leptin or an analog thereof. The XTEN may bechemically attached to the sulfur atom of a cysteine residue located atan internal position of leptin or an analog thereof.

The TA may be Toxin-550 or an analog thereof. The XTEN may be attachedto the N-terminus, C-terminus, or the N- and C-terminus of Toxin-550 oran analog thereof. The XTEN may be attached to a cysteine residuelocated at the N- or C-terminus of Toxin-550 or an analog thereof. TheXTEN may be chemically attached to a cysteine residue located at the N-or C-terminus of Toxin-550 or an analog thereof. The XTEN may beattached to the sulfur atom of a cysteine residue located at the N- orC-terminus of Toxin-550 or an analog thereof. The XTEN may be chemicallyattached to the sulfur atom of a cysteine residue located at the N- orC-terminus of Toxin-550 or an analog thereof. The XTEN may be attachedto an internal residue of Toxin-550 or an analog thereof. The XTEN maybe attached to a cysteine residue located at an internal position ofToxin-550 or an analog thereof. The XTEN may be chemically attached to acysteine residue located at an internal position of Toxin-550 or ananalog thereof. The XTEN may be attached to the sulfur atom of acysteine residue located at an internal position of Toxin-550 or ananalog thereof. The XTEN may be chemically attached to the sulfur atomof a cysteine residue located at an internal position of Toxin-550 or ananalog thereof.

The XTEN-mTAs disclosed herein may further comprise one or more linkers.The XTEN-mTAs disclosed herein may further comprise two or more linkers.The XTEN-mTAs disclosed herein may further comprise three or morelinkers. The XTEN-mTAs disclosed herein may further comprise four ormore linkers. The XTEN-mTAs disclosed herein may further comprise fiveor more linkers.

Each of the one or more linkers on the XTEN-mTAs may be formed from areactive precursor comprising a halogen atom. The reactive precursor maybe a haloacetamide attached to the XTEN. The haloacetamide may belocated at the N-terminus of the XTEN. The haloacetamide may bechloroacetamide, bromoacetamide, or iodoacetamide.

The TAs disclosed herein may further comprise one or more linkers. TheTAs disclosed herein may further comprise two or more linkers. The TAsdisclosed herein may further comprise three or more linkers. The TAsdisclosed herein may further comprise four or more linkers. The TAsdisclosed herein may further comprise five or more linkers.

The one or more linkers may enable attachment of an XTEN to atherapeutic agent or to another XTEN.

Therapeutic Agent (TA)

The mTAs disclosed herein may comprise one or more therapeutic agents.The mTAs may comprise two or more therapeutic agents. The mTAs maycomprise 3, 4, 5, 6, 7 or more therapeutic agents. The therapeuticagents may be different. The therapeutic agents may be the same. As usedherein, the term “therapeutic agent” refers to candidate proteins orpeptides that modulate the activity of a target protein, target peptide,target cell or target tissue. Modulating the activity can compriseincreasing, decreasing, stimulating, or preventing the activity orexpression of the target protein, peptide, cell or tissue. Targetproteins or peptides include proteins involved in the etiology of adisease or disorder by virtue of expression or activity. TAs maycomprise at least a portion of a protein, biomolecule, chemical, toxin,drug or any combination thereof. Exemplary TAs may include, but are notlimited to, at least a portion of a hormone, kinase, receptor, ligand,growth factor, regulatory protein, metabolic protein, cytokine,chemokine, interferon, phosphatase, antibody or any combination thereof.The TA may be a wild-type peptide or a modified peptide comprising oneor more amino acid additions, deletions, substitutions, or a combinationthereof. The one or more amino acid additions or substitutions may belocated at the C-terminus of the peptide. The one or more amino acidadditions or substitutions may be located at the N-terminus of thepeptide. The one or more amino acid additions or substitutions may belocated at the N-terminus or the C-terminus of the peptide. The one ormore amino acid additions or substitutions may be located at both theN-terminus and C-terminus of the peptide. The one or more amino acidadditions or substitutions may be located at a non-terminus position ofthe peptide.

The TA may be a hormone. Examples of hormones include, but are notlimited to, peptide hormones, lipid and phospholipid-derived hormones,and monoamines. Peptide hormones generally consist of chains of aminoacids. Examples of small peptide hormones include, but are not limitedto thyrotropin-releasing hormone (TRH) and vasopressin. Peptidescomposed of scores or hundreds of amino acids may be referred to asproteins. Examples of protein hormones include insulin and growthhormone. More complex protein hormones may bear carbohydrate side-chainsand may be called glycoprotein hormones. Luteinizing hormone,follicle-stimulating hormone and thyroid-stimulating hormone areexamples of glycoprotein hormones. Lipid and phospholipid-derivedhormones are generally derived from lipids such as linoleic acid andarachidonic acid and phospholipids. Protein hormones may comprisesteroid hormones that are derived from cholesterol and the eicosanoids.Examples of steroid hormones are testosterone and cortisol. Eicosanoidsmay comprise prostaglandins. Monoamines may be derived from aromaticamino acids like phenylalanine, tyrosine, tryptophan by the action ofaromatic amino acid decarboxylase enzymes. The TA may be oxyntomodulin.The TA may be exendin-4. The TA may be exenatide. The TA may beglucagon-like peptide (GLP-1). The TA may be glucagon. The TA may beleptin. The TA may be betatrophin.

The TA may be a relaxin peptide or derivative thereof. The relaxinpeptide may comprise a modified relaxin peptide. The modified relaxinpeptide may comprise at least a portion of a wild-type relaxin peptidecomprising one or more amino acid mutations. The relaxin peptide maycomprise at least a portion of an A chain and/or B chain of a relaxinpeptide. The relaxin peptide may comprise one or more amino acidmutations. The one or more amino acid mutations may comprise a deletion,substitution, addition or a combination thereof. The one or more aminoacid mutations may comprise addition of one or more amino acid residuesto the wild-type relaxin polypeptide. The one or more amino acidmutations may comprise substitution of one or more amino acid residuesof the wild-type relaxin polypeptide. The one or more amino acidmutations may comprise deletion of one or more amino acid residues ofthe wild-type relaxin polypeptide. The one or more amino acid mutationsmay comprise one or more amino acid substitutions of one or more aminoacid residues in an A chain and/or B chain of a wild-type relaxinpeptide. The one or more amino acid mutations may comprise one or moreamino acid substitutions of one or more amino acid residues in an Achain of a wild-type relaxin peptide. The one or more amino acidsubstitutions of one or more amino acid residues in the A chain may beselected from a group consisting of Y3C, A7C, T16C, R18C, S19C, or acombination thereof. The one or more amino acid mutations may compriseone or more amino acid substitutions of one or more amino acid residuesin a B chain of a wild-type relaxin peptide. The one or more amino acidsubstitutions of one or more amino acid residues in the B chain may beselected from a group consisting of D1A, S2C, M4C, S26C, and S29C, orany combination thereof. The one or more amino acid mutations maycomprise a Y3C substitution in an A chain of a wild-type relaxinpeptide. The one or more amino acid mutations may comprise an A7Csubstitution in an A chain of a wild-type relaxin peptide. The one ormore amino acid mutations may comprise a T16C substitution in an A chainof a wild-type relaxin peptide. The one or more amino acid mutations maycomprise a R18C substitution in an A chain of a wild-type relaxinpeptide. The one or more amino acid mutations may comprise a S19Csubstitution in an A chain of a wild-type relaxin peptide. The one ormore amino acid mutations may comprise a S2C substitution in a B chainof a wild-type relaxin peptide. The one or more amino acid mutations maycomprise a DIA substitution in a B chain of a wild-type relaxin peptide.The one or more amino acid mutations may comprise a M4C substitution ina B chain of a wild-type relaxin peptide. The one or more amino acidmutations may comprise a S26C substitution in a B chain of a wild-typerelaxin peptide. The one or more amino acid mutations may comprise aS29C substitution in a B chain of a wild-type relaxin peptide. The oneor more amino acid mutations may comprise substituting one or more aminoacid residues of a wild-type relaxin peptide with a cysteine residue.The one or more amino acid residues of the wild-type relaxin peptide areselected from a group consisting of alanine, methionine, arginine,serine, threonine, and tyrosine. The one or more amino acid mutationsmay comprise adding one or more amino acid residues to a wild-typerelaxin peptide.

The TA may be a growth factor. Growth factors may include, but are notlimited to, cytokines and hormones. Examples of growth factors include,but are not limited to, adrenomedullin (AM), angiopoietin (Ang),autocrine motility factor, bone morphogenetic proteins (BMPs),brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF),erythropoietin (EPO), fibroblast growth factor (FGF), glial cellline-derived neurotrophic factor (GDNF), granulocyte colony-stimulatingfactor (G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growthfactor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growthfactor (IGF), migration-stimulating factor, myostatin (GDF-8), nervegrowth factor (NGF) and other neurotrophins, platelet-derived growthfactor (PDGF), thrombopoietin (TPO), transforming growth factor alpha(TGF-α), transforming growth factor beta (TGF-β), tumor necrosisfactor-alpha (TNF-α) and vascular endothelial growth factor (VEGF). TheTA may be fibroblast growth factor 21 (FGF21).

The TA may be a cell regulatory protein. The TA may be a cell regulatoryprotein of the transforming growth factor beta superfamily. The TA maybe a member of the decapentaplegic-Vg related (DVR) related subfamily.The TA may be a member of the activin/inhibin subfamily. The TA may be amember of the TGF-beta subfamily. The TA may be a growth differentiationfactor (GDF). The GDF may be GDF1, GDF2, GDF3, GDF5, GDF6, GFD8, GDF9,GDF10, GDF11, and GDF15. The TA may be growth differentiation factor 11(GDF11).

The TA may be a protein. The protein may be a member of theangiopoietin-like family of secreted factors. The protein may be anangiopoietin-like protein (ANGPTL). Examples of ANGPTLs include, but arenot limited to, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6 andANGPTL7. The TA may be ANGPTL3.

The TA may comprise a peptide selected from relaxin, H1 relaxin, H2relaxin, H3 relaxin, human INSL3, human INSL4, human INSL6, human IGF1,human IGFII, human insulin, oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, leptin, betatrophin, FGF 21,GDF 11, ANGPTL3, peptide-based toxin, Moka, and VM-24, and derivativesthereof, the derivative being a peptide comprising one or more aminoacid additions, deletions, or substitutions, or a combination thereof.The TA may comprise relaxin, oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, leptin, betatrophin, FGF 21,GDF 11, ANGPTL3, peptide-based toxin, Moka, or VM-24, or a derivativethereof, the derivative being a peptide comprising one or more aminoacid additions, deletions, or substitutions, or a combination thereof.The TA may comprise oxyntomodulin, exenatide, exendin-4, glucagon-likeprotein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, or aGLP-1R and GCGR dual agonist, or a derivative thereof, the derivativebeing a peptide comprising one or more amino acid additions, deletions,or substitutions, or a combination thereof. The TA may comprise H1relaxin, H2 relaxin, H3 relaxin, human INSL3, human INSL4, human INSL6,human IGF1, human IGFII, or human insulin, or a derivative thereof, thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof. The TA maycomprise H1 relaxin, H2 relaxin, or H3 relaxin, or a derivative thereof,the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. The TAmay comprise human INSL3, human INSL4, human INSL6, human IGF1, or humanIGFII, or a derivative thereof, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The TA may comprise a peptidyltoxin or a derivative thereof, the derivative being a peptide comprisingone or more amino acid additions, deletions, or substitutions, or acombination thereof. The TA may comprise Toxin-550, Moka, or VM-24, or aderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof. The TA may comprise human insulin or a derivative thereof, thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof.

The TA may be encoded by a nucleotide sequence based on or derived froma nucleotide sequence selected from the group comprising SEQ ID NO: 1-9.The TA may be encoded by a nucleotide sequence that is at least about50% homologous to a nucleotide sequence selected from the groupcomprising SEQ ID NO: 1-9. The TA may be encoded by a nucleotidesequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,92%, 95%, 97%, 99%, or 100% homologous to a nucleotide sequence selectedfrom the group comprising SEQ ID NO: 1-9. The TA may be encoded by anucleotide sequence that is at least about 70% homologous to anucleotide sequence selected from the group comprising SEQ ID NO: 1-9.The TA may be encoded by a nucleotide sequence that is at least about75% homologous to a nucleotide sequence selected from the groupcomprising SEQ ID NO: 1-9. The TA may be encoded by a nucleotidesequence that is at least about 80% homologous to a nucleotide sequenceselected from the group comprising SEQ ID NO: 1-9. The TA may compriseone or more nucleotide sequences selected from the group comprising SEQID NO: 1-9. The TA may comprise two or more nucleotide sequencesselected from the group comprising SEQ ID NO: 1-9. The TA may comprisethree or more nucleotide sequences selected from the group comprisingSEQ ID NO: 1-9. The TA may comprise four or more nucleotide sequencesselected from the group comprising SEQ ID NO: 1-9. The TA may comprise5, 6, 7, 8, or 9 nucleotide sequences selected from the group comprisingSEQ ID NO: 1-9.

The TA may comprise an amino acid sequence selected from the groupcomprising SEQ ID NO: 10-56. The TA may comprise an amino acid sequencethat is at least about 50% homologous to an amino acid sequence selectedfrom the group comprising SEQ ID NO: 10-56. The TA may comprise an aminoacid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 99%, or 100% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 10-56. The TA may comprisean amino acid sequence that is at least about 70% homologous to an aminoacid sequence selected from the group comprising SEQ ID NO: 10-56. TheTA may comprise an amino acid sequence that is at least about 75%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 10-56. The TA may comprise an amino acid sequence that is atleast about 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 10-56. The TA may comprise one or more aminoacid sequences selected from the group comprising SEQ ID NO: 10-56. TheTA may comprise two or more amino acid sequences selected from the groupcomprising SEQ ID NO: 10-56. The TA may comprise three or more aminoacid sequences selected from the group comprising SEQ ID NO: 10-56. TheTA may comprise four or more amino acid sequences selected from thegroup comprising SEQ ID NO: 10-56. The TA may comprise 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 or more amino acid sequences selected from thegroup comprising SEQ ID NO: 10-56.

The TA may comprise 20 or more consecutive amino acids from an aminoacid sequence selected from the group comprising SEQ ID NO: 10-56. TheTA may comprise 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or moreconsecutive amino acids from an amino acid sequence selected from thegroup comprising SEQ ID NO: 10-56.

The TAs may be from a mammal or non-mammal. The TAs may be from a human.

Alternatively, the TAs may be from a goat, sheep, cow, rabbit, monkey,dog, cat or a combination thereof. The TAs may be from a reptile. TheTAs may be from a snake or lizard. The TAs may be from an amphibian. TheTAs may be from a frog or toad. The TAs may be from an avian. The TAsmay be recombinant.

The TAs disclosed herein may further comprise one or more linkers. TheTAs disclosed herein may further comprise two or more linkers. The TAsdisclosed herein may further comprise three or more linkers. The TAsdisclosed herein may further comprise four or more linkers. The TAsdisclosed herein may further comprise five or more linkers.

Pharmacokinetics

Mechanisms by which the modified therapeutic agents positively influencepharmacokinetic or pharmacodynamic behavior include, but are not limitedto, (i) preventing or mitigating in vivo proteolytic degradation orother activity-diminishing chemical modification of the therapeuticagent; (ii) improving half-life or other pharmacokinetic properties byreducing renal filtration, decreasing receptor-mediated clearance orincreasing bioavailability; (iii) reducing toxicity; (iv) improvingsolubility; and/or (v) increasing biological activity and/or targetselectivity of the therapeutic agent.

The half-life extending moieties may enhance one or more pharmacokineticproperties of a therapeutic agent (TA) when attached to the TA.

The modified therapeutic agents disclosed herein may enhance the one ormore pharmacokinetic properties of the TA by at least about 200% asmeasured by pharmacodynamics when compared to the TA alone. The modifiedtherapeutic agents disclosed herein may enhance the one or morepharmacokinetic properties of the TA by at least about 300%, 400%, 500%,600%, 700%, 800%, 900%, 1000% as measured by pharmacodynamics whencompared to the TA alone. The modified therapeutic agents disclosedherein may enhance the one or more pharmacokinetic properties of the TAby at least about 250% as measured by pharmacodynamics when compared tothe TA alone. The modified therapeutic agents disclosed herein mayenhance the one or more pharmacokinetic properties of the TA by at leastabout 300% as measured by pharmacodynamics when compared to the TAalone. The modified therapeutic agents disclosed herein may enhance theone or more pharmacokinetic properties of the TA by at least about 350%as measured by pharmacodynamics when compared to the TA alone. Themodified therapeutic agents disclosed herein may enhance the one or morepharmacokinetic properties of the TA by at least about 400% as measuredby pharmacodynamics when compared to the TA alone. The modifiedtherapeutic agents disclosed herein may enhance the one or morepharmacokinetic properties of the TA by at least about 500% as measuredby pharmacodynamics when compared to the TA alone.

The pharmacokinetic properties may comprise a half-life. The half-lifeof the modified therapeutic agent may be at least about two-fold longercompared to the half-life of the TA alone. The half-life of the modifiedtherapeutic agent disclosed herein may be at least about 3-fold, 4-fold,or 5-fold longer compared to the half-life of the TA alone. Thehalf-life of the modified therapeutic agent disclosed herein may be atleast about 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or50-fold longer compared to the half-life of the TA alone. The half-lifeof the modified therapeutic agent disclosed herein may be at least about5-fold longer compared to the half-life of the TA alone. The half-lifeof the modified therapeutic agent disclosed herein may be at least about10-fold longer compared to the half-life of the TA alone.

In addition, the modified therapeutic agents may have positive effectson terms of increasing manufacturability, and/or reducing immunogenicityof the therapeutic agentcompared to an unconjugated form of thetherapeutic agent.

A modified therapeutic agent may have comparable activity to the TAalone. A modified therapeutic agent may have similar activity to the TAalone. A modified therapeutic agent may have increased activity to theTA alone. A modified therapeutic agent may have decreased activity tothe TA alone.

Attachment of one or more lipids to a TA to form an LC may diminish theactivity of the LC relative to the TA alone by no more than 2-, 3-, 4-,5-, 6-, 7-, 8-, 9-, or 10-fold less activity. Attachment of a polyglycolregion to a TA to form a modified therapeutic agent may diminish theactivity of the modified therapeutic agent relative to the TA alone byno more than 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold less activity.

Therapeutic Use

Further disclosed herein are mTAs for treating, alleviating, inhibitingand/or preventing one or more diseases and/or conditions. The diseaseand/or condition may be a chronic disease or condition. Alternatively,the disease and/or condition may be an acute disease or condition. Thedisease or condition may be recurrent, refractory, accelerated, or inremission. The disease or condition may affect one or more cell types.The one or more diseases and/or conditions may be an autoimmune disease,inflammatory disease, cardiovascular disease and pregnancy. The mTAsdisclosed herein may be administered to a subject in need thereof.

Disclosed herein are methods of treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject a composition comprising an LC, wherein the LC comprises a lipidattached to a therapeutic agent, wherein the therapeutic agent isrelaxin or a derivative thereof. The disease or condition may be acardiovascular disease. The cardiovascular disease may be acute heartfailure. Additional cardiovascular diseases include, but are not limitedto, congestive heart failure, compensated heart failure or decompensatedheart failure. The disease or condition may be an autoimmune disorder.The autoimmune disorder may be scleroderma, diffuse scleroderma orsystemic scleroderma. The disease or condition may be an inflammatorydisease. The inflammatory disease may be fibromyalgia. The disease orcondition may be fibrosis. Alternatively, the disease or condition maybe pregnancy. The LC may be used to treat preeclampsia or induce labor.The LC may be administered with one or more additional therapeuticagents. The additional therapeutic agents may comprise one or more ofanti-inflammatory drugs, statins, diuretics, beta-blockers, angiotensinconverting enzyme inhibitors and angiotensin II receptor blockers. Theadditional therapeutic agent may be aspirin.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more LCs, wherein the one or more lipid conjugates (LCs)comprise (a) one or more lipids, the lipids selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more lipids are attached to at least one cysteine residue in the oneor more therapeutic agents. The one or more TAs may comprise relaxin.The disease or condition may be a cardiovascular disease. Thecardiovascular disease may be acute heart failure. Additionalcardiovascular diseases include, but are not limited to, congestiveheart failure, compensated heart failure or decompensated heart failure.The disease or condition may be an autoimmune disorder. The autoimmunedisorder may be scleroderma, diffuse scleroderma or systemicscleroderma. The disease or condition may be an inflammatory disease.The inflammatory disease may be fibromyalgia. The disease or conditionmay be fibrosis. Alternatively, the disease or condition is pregnancy.The LC may be used to treat preeclampsia or induce labor. The LC may beadministered with one or more additional therapeutic agents. Theadditional therapeutic agents may comprise one or more ofanti-inflammatory drugs, statins, diuretics, beta-blockers, angiotensinconverting enzyme inhibitors and angiotensin II receptor blockers. Theadditional therapeutic agent may be aspirin.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more LCs, wherein the one or more lipid conjugates (LCs)comprise (a) one or more lipids, the lipids selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof; and (b) a therapeutic agent(TA), wherein TA is a peptide and the one or more lipids are attached tothe peptide via an amino acid on the peptide. The TA may compriserelaxin. The disease or condition may be a cardiovascular disease. Thecardiovascular disease may be acute heart failure. Additionalcardiovascular diseases include, but are not limited to, congestiveheart failure, compensated heart failure or decompensated heart failure.The disease or condition may be an autoimmune disorder. The autoimmunedisorder may be scleroderma, diffuse scleroderma or systemicscleroderma. The disease or condition may be an inflammatory disease.The inflammatory disease may be fibromyalgia. The disease or conditionmay be fibrosis. Alternatively, the disease or condition is pregnancy.The LC may be used to treat preeclampsia or induce labor. The LC may beadministered with one or more additional therapeutic agents. Theadditional therapeutic agents may comprise one or more ofanti-inflammatory drugs, statins, diuretics, beta-blockers, angiotensinconverting enzyme inhibitors and angiotensin II receptor blockers. Theadditional therapeutic agent may be aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more lipid conjugates (LCs) of Formula (Ia): TA-A¹-P¹-L,wherein TA is a therapeutic agent; A¹ is a chemical group linking TA andP¹; P¹ is a bond or -PEG-A²-; PEG is a chemical group comprising one ormore polyethyleneglycol subunits; A² is a chemical group linking PEG andL; and L is a lipid. The TA may comprise relaxin. The disease orcondition may be a cardiovascular disease. The cardiovascular diseasemay be acute heart failure. Additional cardiovascular diseases include,but are not limited to, congestive heart failure, compensated heartfailure or decompensated heart failure. The disease or condition may bean autoimmune disorder. The autoimmune disorder may be scleroderma,diffuse scleroderma or systemic scleroderma. The disease or conditionmay be an inflammatory disease. The inflammatory disease may befibromyalgia. The disease or condition may be fibrosis. Alternatively,the disease or condition is pregnancy. The LC may be used to treatpreeclampsia or induce labor. The LC may be administered with one ormore additional therapeutic agents. The additional therapeutic agentsmay comprise one or more of anti-inflammatory drugs, statins, diuretics,beta-blockers, angiotensin converting enzyme inhibitors and angiotensinII receptor blockers. The additional therapeutic agent may be aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more lipid conjugates (LCs) of Formula (I): TA-A¹-P¹-L,wherein TA is a therapeutic agent; A¹ is a chemical group linking TA andP¹ or L; P¹ is a bond or comprises polyglycol; and L is a lipid. The TAmay comprise relaxin. The disease or condition may be a cardiovasculardisease. The cardiovascular disease may be acute heart failure.Additional cardiovascular diseases include, but are not limited to,congestive heart failure, compensated heart failure or decompensatedheart failure. The disease or condition may be an autoimmune disorder.The autoimmune disorder may be scleroderma, diffuse scleroderma orsystemic scleroderma. The disease or condition may be an inflammatorydisease. The inflammatory disease may be fibromyalgia. The disease orcondition may be fibrosis. Alternatively, the disease or condition ispregnancy. The LC may be used to treat preeclampsia or induce labor. TheLC may be administered with one or more additional therapeutic agents.The additional therapeutic agents may comprise one or more ofanti-inflammatory drugs, statins, diuretics, beta-blockers, angiotensinconverting enzyme inhibitors and angiotensin II receptor blockers. Theadditional therapeutic agent may be aspirin.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more LCs, wherein the one or morelipid conjugates (LCs) comprise (a) one or more lipids, the lipidsselected from a group consisting of sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols; and (b) one or more therapeutic agents (TA), whereinthe one or more lipids are attached to the one or more cysteine residuesin the one or more therapeutic agents. The one or more TAs may compriseGLP-1, Exendin-4, exenatide, oxyntomodulin, glucagon, FGF21, orderivative thereof. The GLP-1 may be a human GLP-1. The FGF21 may be ahuman FGF21. The metabolic disease or condition may be diabetes. Themetabolic disease or condition may be glycogen storage disease,phenylketonuria, maple syrup urine disease, glutaric acidemia type 1,Carbamoyl phosphate synthetase I deficiency, alcaptonuria, Medium-chainacyl-coenzyme A dehydrogenase deficiency (MCADD), acute intermittentporphyria, Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia,congenital adrenal hyperplasia, Kearns-Sayre syndrome, Zellwegersyndrome, Gaucher's disease, or Niemann Pick disease.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more LCs, wherein the one or morelipid conjugates (LCs) comprise (a) one or more lipids, the lipidsselected from a group consisting of sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,fatty amines, and fatty alcohols, and derivatives thereof and (b) atherapeutic agent (TA), wherein the TA is a peptide and the one or morelipids are attached to the TA via an amino acid residue on the peptide.The TA may comprise GLP-1, Exendin-4, exenatide, oxyntomodulin,glucagon, FGF21, or derivative thereof. The GLP-1 may be a human GLP-1.The FGF21 may be a human FGF21. The metabolic disease or condition maybe diabetes. The metabolic disease or condition may be glycogen storagedisease, phenylketonuria, maple syrup urine disease, glutaric acidemiatype 1, Carbamoyl phosphate synthetase I deficiency, alcaptonuria,Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), acuteintermittent porphyria, Lesch-Nyhan syndrome, lipoid congenital adrenalhyperplasia, congenital adrenal hyperplasia, Kearns-Sayre syndrome,Zellweger syndrome, Gaucher's disease, or Niemann Pick disease.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more lipid conjugates (LCs) ofFormula (Ia): TA-A¹-P¹-L, wherein TA is a therapeutic agent; A¹ is achemical group linking TA and P¹; P¹ is a bond or -PEG-A²-; PEG is achemical group comprising one or more polyethyleneglycol subunits; A² isa chemical group linking PEG and L; and L is a lipid. The TA maycomprise GLP-1, Exendin-4, exenatide, oxyntomodulin, glucagon, FGF21, orderivative thereof. The GLP-1 may be a human GLP-1. The FGF21 may be ahuman FGF21. The one or more lipids may comprise one or more sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols. The metabolic disease orcondition may be diabetes. The metabolic disease or condition may beglycogen storage disease, phenylketonuria, maple syrup urine disease,glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency,alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency(MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia,Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or NiemannPick disease.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more lipid conjugates (LCs) ofFormula (I): TA-A¹-P¹-L, wherein TA is a therapeutic agent; A¹ is achemical group linking TA and P¹ or L; P¹ is a bond or comprisespolyglycol; and L is a lipid. The TA may comprise GLP-1, exendin-4,exenatide, oxyntomodulin, glucagon, FGF21, or derivative thereof. TheGLP-1 may be a human GLP-1. The FGF21 may be a human FGF21. The one ormore lipids may comprise one or more sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols. The metabolic disease or condition may be diabetes.The metabolic disease or condition may be glycogen storage disease,phenylketonuria, maple syrup urine disease, glutaric acidemia type 1,Carbamoyl phosphate synthetase I deficiency, alcaptonuria, Medium-chainacyl-coenzyme A dehydrogenase deficiency (MCADD), acute intermittentporphyria, Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia,congenital adrenal hyperplasia, Kearns-Sayre syndrome, Zellwegersyndrome, Gaucher's disease, or Niemann Pick disease.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more LCs, wherein the one or morelipid conjugates (LCs) comprise (a) one or more lipids, the lipidsselected from a group consisting of sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols; and (b) one or more therapeutic agents (TAs),wherein the one or more lipids are attached to at least one cysteineresidue in the one or more therapeutic agents. The one or more TAs maycomprise GLP-1, exendin-4, exenatide, oxyntomodulin, glucagon, orderivative thereof. The GLP-1 may be a human GLP-1. The CNS disorder maybe Alzheimer's disease (AD). Additional CNS disorders include, but arenot limited to, encephalitis, meningitis, tropical spastic paraparesis,arachnoid cysts, Huntington's disease, locked-in syndrome, Parkinson'sdisease, Tourette's, and multiple sclerosis.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more LCs, wherein the one or morelipid conjugates (LCs) comprise (a) one or more lipids, the lipidsselected from a group consisting of sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,fatty amines, and fatty alcohols, and derivatives thereof and (b) atherapeutic agent (TA), wherein the TA is a peptide and the one or morelipids are attached to the TA via an amino acid residue on the peptide.The TA may comprise GLP-1, exendin-4, exenatide, oxyntomodulin,glucagon, or derivative thereof. The GLP-1 may be a human GLP-1. The CNSdisorder may be Alzheimer's disease (AD). Additional CNS disordersinclude, but are not limited to, encephalitis, meningitis, tropicalspastic paraparesis, arachnoid cysts, Huntington's disease, locked-insyndrome, Parkinson's disease, Tourette's, and multiple sclerosis.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more lipid conjugates (LCs) ofFormula (Ia): TA-A¹-P¹-L, wherein TA is a therapeutic agent; A¹ is achemical group linking TA and P¹; P¹ is a bond or -PEG-A²-; PEG is achemical group comprising one or more polyethyleneglycol subunits; A² isa chemical group linking PEG and L; and L is a lipid. The one or morelipids may comprise one or more sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols. The one or more lipids may comprise one or more sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols. The TA may compriseGLP-1, exendin-4, exenatide, oxyntomodulin, glucagon, or derivativethereof. The GLP-1 may be a human GLP-1. The CNS disorder may beAlzheimer's disease (AD). Additional CNS disorders include, but are notlimited to, encephalitis, meningitis, tropical spastic paraparesis,arachnoid cysts, Huntington's disease, locked-in syndrome, Parkinson'sdisease, Tourette's, and multiple sclerosis.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more lipid conjugates (LCs) ofFormula (I): TA-A¹-P¹-L, wherein TA is a therapeutic agent; A¹ is achemical group linking TA and P¹ or L; P¹ is a bond or comprisespolyglycol; and L is a lipid. The one or more lipids may comprise one ormore sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols. The oneor more lipids may comprise one or more sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, or fatty alcohols, or derivatives thereof. The TA may compriseGLP-1, exendin-4, exenatide, oxyntomodulin, glucagon, or derivativethereof. The GLP-1 may be a human GLP-1. The CNS disorder may beAlzheimer's disease (AD). Additional CNS disorders include, but are notlimited to, encephalitis, meningitis, tropical spastic paraparesis,arachnoid cysts, Huntington's disease, locked-in syndrome, Parkinson'sdisease, Tourette's, and multiple sclerosis.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more LCs, wherein the one or more lipid conjugates (LCs)comprise (a) one or more lipids, the lipids selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols; and (b) one or more therapeutic agents (TAs), wherein the oneor more lipids are attached to at least one cysteine residue in the oneor more therapeutic agents. The one or more TAs may comprise GLP-1,exendin-4, exenatide, oxyntomodulin, glucagon, or derivative thereof.The GLP-1 may be a human GLP-1. The disease or condition may be ametabolic disease or disorder. The disease or condition may be diabetes.The disease or condition may be obesity. Additional diseases and/orconditions which benefit from a GLP-1R and/or GCGR agonist include, butare not limited to, dyslipidemia, cardiovascular and fatty liverdiseases.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more LCs, wherein the one or more lipid conjugates (LCs)comprise (a) one or more lipids, the lipids selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof and (b) a therapeutic agent(TA), wherein the TA is a peptide and the one or more lipids areattached to the TA via an amino acid residue on the peptide. The TA maycomprise GLP-1, exendin-4, exenatide, oxyntomodulin, glucagon, orderivative thereof. The GLP-1 may be a human GLP-1. The disease orcondition may be a metabolic disease or disorder. The disease orcondition may be diabetes. The disease or condition may be obesity.Additional diseases and/or conditions which benefit from a GLP-1R and/orGCGR agonist include, but are not limited to, dyslipidemia,cardiovascular and fatty liver diseases.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more lipid conjugates (LCs) of Formula (Ia): TA-A¹-P¹-L,wherein TA is a therapeutic agent; A¹ is a chemical group linking TA andP¹; P¹ is a bond or -PEG-A²-; PEG is a chemical group comprising one ormore polyethyleneglycol subunits; A² is a chemical group linking PEG andL; and L is a lipid. The one or more lipids may comprise one or moresterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, and fatty alcohols. The one or morelipids may comprise one or more sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols. The TA may comprise GLP-1, exendin-4, exenatide,oxyntomodulin, glucagon, or derivative thereof. The GLP-1 may be a humanGLP-1. The disease or condition may be a metabolic disease or disorder.The disease or condition may be diabetes. The disease or condition maybe obesity. Additional diseases and/or conditions which benefit from aGLP-1R and/or GCGR agonist include, but are not limited to,dyslipidemia, cardiovascular and fatty liver diseases.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more lipid conjugates (LCs) of Formula (I): TA-A¹-P¹-L,wherein TA is a therapeutic agent; A¹ is a chemical group linking TA andP¹ or L; P¹ is a bond or comprises polyglycol; and L is a lipid. The oneor more lipids may comprise one or more sterols, sterol derivatives,bile acids, vitamin E derivatives, fatty di-acids, fatty acids, fattyamides, and fatty alcohols, or derivatives thereof. The one or morelipids may comprise one or more sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, orfatty alcohols, or derivatives thereof. The TA may comprise GLP-1,exendin-4, exenatide, oxyntomodulin, glucagon, or derivative thereof.The GLP-1 may be a human GLP-1. The disease or condition may be ametabolic disease or disorder. The disease or condition may be diabetes.The disease or condition may be obesity. Additional diseases and/orconditions which benefit from a GLP-1R and/or GCGR agonist include, butare not limited to, dyslipidemia, cardiovascular and fatty liverdiseases.

Further disclosed herein are mTAs for use in treating, alleviating,inhibiting and/or preventing one or more diseases and/or conditions. Thedisease and/or condition may be a chronic disease or condition.Alternatively, the disease and/or condition may be an acute disease orcondition. The disease or condition may be recurrent, refractory,accelerated, or in remission. The disease or condition may affect one ormore cell types. The one or more diseases and/or conditions may be anautoimmune disease, inflammatory disease, cardiovascular disease and/orpregnancy. The modified therapeutic agents (mTAs) may comprise atherapeutic agent and one or more half-life extending moieties, whereinthe therapeutic agent is a peptide that is covalently attached to eachof the one or more half-life extending moieties via a cysteine residueon the peptide; and the half-life of the modified therapeutic agent islonger than the half-life of the peptide alone. The mTAs may comprisetwo or more half-life extending moieties. The two or more half-lifeextending moieties may be identical. The two or more half-life extendingmoieties may be different. Each of the one or more half-life extendingmoieties may comprise a lipid, a polyglycol region, or a combinationthereof. Each of the one or more half-life extending moieties maycomprise a lipid. Each of the one or more half-life extending moietiesmay comprise a polyglycol region. Each of the one or more half-lifeextending moieties may comprise a lipid and a polyglycol region. Each ofthe one or more half-life extending moieties may comprise an extendedrecombinant polypeptide (XTEN) comprising (i) an amino acid sequencecharacterized in that the sum of glycine (G), alanine (A), serine (S),threonine (T), glutamate (E), aspartate (D), leucine (L) and proline (P)residues constitutes more than about 70% of the total amino acidsequence; (ii) a substantially non-repetitive amino acid sequence; (iii)an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets. The XTEN may comprise an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%,97%, or 99% the total amino acid sequence. The XTEN may comprise anamino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 75%,77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% the total amino acidsequence. The XTEN may comprise an amino acid sequence selected from SEQID NOs: 66-67. The XTEN may comprise an amino acid sequence that is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%,95%, 97%, or 99% or more homologous to an amino acid sequence of SEQ IDNOs: 66-67. The peptide may comprise one or more amino acid additions,deletions, or substitutions, or a combination thereof. The peptide maybe selected from relaxin, H1 relaxin, H2 relaxin, H3 relaxin, humanINSL3, human INSL4, human INSL6, human IGF1, human IGFII, human insulin,oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1 (GLP-1),GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, leptin, betatrophin, FGF 21, GDF 11, ANGPTL3, peptide-basedtoxin, Moka, and VM-24, or a derivative thereof, the derivative being apeptide comprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The peptide may be selectedfrom relaxin, H1 relaxin, H2 relaxin, H3 relaxin, human INSL3, humanINSL4, human INSL6, human IGF1, human IGFII, human insulin,oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1 (GLP-1),GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, leptin, betatrophin, FGF 21, GDF 11, ANGPTL3, peptide-basedtoxin, Moka, and VM-24, or a derivative thereof, the derivative being apeptide comprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The peptide may be relaxin or aderivative thereof, the derivative being a peptide comprising one ormore amino acid additions, deletions, or substitutions, or a combinationthereof. The peptide may be encoded by an amino acid sequence comprisingat least a portion of a polypeptide sequence selected from a groupconsisting of SEQ ID NO: 10-56. The peptide may be encoded by an aminoacid sequence comprising 10 or more amino acids based on or derived froma polypeptide sequence selected from a group consisting of SEQ ID NO:10-56. The peptide may comprise an amino acid sequence that is at leastabout 50% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 10-56. The peptide may comprise an amino acidsequence that is at least 80% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 10-56. The peptide maycomprise one or more amino acid sequences selected from the groupcomprising SEQ ID NO: 10-56. The peptide may comprise two or more aminoacid sequences selected from the group comprising SEQ ID NO: 10-56. Thepeptide may further comprise a linker. The cysteine residue may belocated on the N-terminus or C-terminus of the peptide. The cysteineresidue may be located on a non-terminus position of the peptide. Thecysteine residue may be an amino acid addition or substitution on thepeptide. The mTAs disclosed herein may be administered to a subject inneed thereof. The mTA may be an XTEN-mTA. The mTA may be a LC.

Disclosed herein are methods of treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject a composition comprising an mTA, wherein the mTA comprises ahalf-life extending moiety attached to a therapeutic agent, wherein thetherapeutic agent is relaxin or a derivative thereof. The XTEN maycomprise an amino acid sequence characterized in that the sum of glycine(G), alanine (A), serine (S), threonine (T), glutamate (E), aspartate(D), leucine (L) and proline (P) residues constitutes more than about75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% the total aminoacid sequence. The XTEN may comprise an amino acid sequence selectedfrom SEQ ID NOs: 66-67. The XTEN may comprise an amino acid sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%,90%, 93%, 95%, 97%, or 99% or more homologous to an amino acid sequenceof SEQ ID NOs: 66-67. The therapeutic agent may further comprise alinker. The disease or condition may be a cardiovascular disease. Thecardiovascular disease may be acute heart failure. Additionalcardiovascular diseases include, but are not limited to, congestiveheart failure, compensated heart failure or decompensated heart failure.The disease or condition may be an autoimmune disorder. The autoimmunedisorder may be scleroderma, diffuse scleroderma or systemicscleroderma. The disease or condition may be an inflammatory disease.The inflammatory disease may be fibromyalgia. The disease or conditionmay be fibrosis. Alternatively, the disease or condition may bepregnancy. The mTA may be used to treat preeclampsia or induce labor.The mTA may be administered with one or more additional therapeuticagents. The additional therapeutic agents may comprise one or more ofanti-inflammatory drugs, statins, diuretics, beta-blockers, angiotensinconverting enzyme inhibitors and angiotensin II receptor blockers. Theadditional therapeutic agent may be aspirin. The mTA may be an XTEN-mTA.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more mTAs, wherein the one or more modified therapeuticagents (mTAs) comprise (a) one or more half-life extending moieties, thehalf-life extending moieties comprising an extended recombinantpolypeptide (XTEN) comprising (i) an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 70% of the total amino acid sequence; (ii) asubstantially non-repetitive amino acid sequence; (iii) an amino acidsequence that has less than about 10% alpha helices; and/or (iv) anamino acid sequence that has less than about 10% beta-sheets; and (b) atherapeutic agents (TA), wherein the one or more half-life extendingmoieties are attached to at least one cysteine residue in the one ormore therapeutic agents. The TA may comprise relaxin. The TA maycomprise a linker. The TA may comprise a modified polypeptide. Themodified polypeptide may comprise a polypeptide comprising one or moremutations. The one or more mutations may be a substitution, deletion, orinsertion. The modified peptide may comprise a polypeptide attached to alinker. The disease or condition may be a cardiovascular disease. Thecardiovascular disease may be acute heart failure. Additionalcardiovascular diseases include, but are not limited to, congestiveheart failure, compensated heart failure or decompensated heart failure.The disease or condition may be an autoimmune disorder. The autoimmunedisorder may be scleroderma, diffuse scleroderma or systemicscleroderma. The disease or condition may be an inflammatory disease.The inflammatory disease may be fibromyalgia. The disease or conditionmay be fibrosis. Alternatively, the disease or condition is pregnancy.The mTA may be used to treat preeclampsia or induce labor. The mTA maybe administered with one or more additional therapeutic agents. Theadditional therapeutic agents may comprise one or more ofanti-inflammatory drugs, statins, diuretics, beta-blockers, angiotensinconverting enzyme inhibitors and angiotensin II receptor blockers. Theadditional therapeutic agent may be aspirin. The mTA may be an XTEN-mTA.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more mTAs, wherein the one or more modified therapeuticagents (mTAs) comprise (a) one or more half-life extending moieties,wherein the one or more half-life extending moieties comprises anextended recombinant polypeptide (XTEN) comprising (i) an amino acidsequence characterized in that the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E), aspartate (D), leucine (L) andproline (P) residues constitutes more than about 70% of the total aminoacid sequence; (ii) a substantially non-repetitive amino acid sequence;(iii) an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets; and (b) a therapeutic agent (TA), wherein TA is a peptideand the one or more half-life extending moieties are attached to thepeptide via an amino acid on the peptide. The XTEN may comprise an aminoacid sequence characterized in that the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E), aspartate (D), leucine (L) andproline (P) residues constitutes more than about 75%, 77%, 80%, 83%,85%, 87%, 90%, 92%, 95%, 97%, or 99% the total amino acid sequence. TheXTEN may comprise an amino acid sequence selected from SEQ ID NOs:66-67. The XTEN may comprise an amino acid sequence that is at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%, 95%,97%, or 99% or more homologous to an amino acid sequence of SEQ ID NOs:66-67. The TA may comprise relaxin. The TA may further comprise alinker. The disease or condition may be a cardiovascular disease. Thecardiovascular disease may be acute heart failure. Additionalcardiovascular diseases include, but are not limited to, congestiveheart failure, compensated heart failure or decompensated heart failure.The disease or condition may be an autoimmune disorder. The autoimmunedisorder may be scleroderma, diffuse scleroderma or systemicscleroderma. The disease or condition may be an inflammatory disease.The inflammatory disease may be fibromyalgia. The disease or conditionmay be fibrosis. Alternatively, the disease or condition is pregnancy.The mTA may be used to treat preeclampsia or induce labor. The mTA maybe administered with one or more additional therapeutic agents. Theadditional therapeutic agents may comprise one or more ofanti-inflammatory drugs, statins, diuretics, beta-blockers, angiotensinconverting enzyme inhibitors and angiotensin II receptor blockers. Theadditional therapeutic agent may be aspirin. The mTA may be an XTEN-mTA.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more modified therapeutic agents (mTAs) of Formula IIa):TA-A¹-P¹-X, wherein TA is a therapeutic agent; A¹ is a chemical grouplinking TA and P¹; P¹ is a bond or -PEG-A²-; PEG is a chemical groupcomprising one or more polyethyleneglycol subunits; A² is a chemicalgroup linking PEG and X; and X is a half-life extending moiety. Thehalf-life extending moiety may comprise an extended recombinantpolypeptide (XTEN) comprising (i) an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 70% of the total amino acid sequence; (ii) asubstantially non-repetitive amino acid sequence; (iii) an amino acidsequence that has less than about 10% alpha helices; and/or (iv) anamino acid sequence that has less than about 10% beta-sheets. The XTENmay comprise an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% thetotal amino acid sequence; and P¹ is a bond. The XTEN may comprise anamino acid sequence selected from SEQ ID NOs: 66-67. The XTEN maycomprise an amino acid sequence that is at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, or 99% or morehomologous to an amino acid sequence of SEQ ID NOs: 66-67. The TA maycomprise relaxin. The TA may further comprise a linker. The disease orcondition may be a cardiovascular disease. The cardiovascular diseasemay be acute heart failure. Additional cardiovascular diseases include,but are not limited to, congestive heart failure, compensated heartfailure or decompensated heart failure. The disease or condition may bean autoimmune disorder. The autoimmune disorder may be scleroderma,diffuse scleroderma or systemic scleroderma. The disease or conditionmay be an inflammatory disease. The inflammatory disease may befibromyalgia. The disease or condition may be fibrosis. Alternatively,the disease or condition is pregnancy. The mTA may be used to treatpreeclampsia or induce labor. The mTA may be administered with one ormore additional therapeutic agents. The additional therapeutic agentsmay comprise one or more of anti-inflammatory drugs, statins, diuretics,beta-blockers, angiotensin converting enzyme inhibitors and angiotensinII receptor blockers. The additional therapeutic agent may be aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more modified therapeutic agents (mTAs) of Formula (II):TA-A¹-P¹-X, wherein TA is a therapeutic agent; A¹ is a chemical grouplinking TA and P¹ or X; P¹ is a bond or comprises polyglycol; and X is ahalf-life extending moiety. The half-life extending moiety may comprisean extended recombinant polypeptide (XTEN) comprising (i) an amino acidsequence characterized in that the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E), aspartate (D), leucine (L) andproline (P) residues constitutes more than about 70% of the total aminoacid sequence; (ii) a substantially non-repetitive amino acid sequence;(iii) an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets. The XTEN may comprise an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%,97%, or 99% the total amino acid sequence; and P¹ is a bond. The XTENmay comprise an amino acid sequence selected from SEQ ID NOs: 66-67. TheXTEN may comprise an amino acid sequence that is at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, or 99%or more homologous to an amino acid sequence of SEQ ID NOs: 66-67. TheTA may comprise relaxin. The TA may further comprise a linker. Thedisease or condition may be a cardiovascular disease. The cardiovasculardisease may be acute heart failure. Additional cardiovascular diseasesinclude, but are not limited to, congestive heart failure, compensatedheart failure or decompensated heart failure. The disease or conditionmay be an autoimmune disorder. The autoimmune disorder may bescleroderma, diffuse scleroderma or systemic scleroderma. The disease orcondition may be an inflammatory disease. The inflammatory disease maybe fibromyalgia. The disease or condition may be fibrosis.Alternatively, the disease or condition is pregnancy. The mTA may beused to treat preeclampsia or induce labor. The mTA may be administeredwith one or more additional therapeutic agents. The additionaltherapeutic agents may comprise one or more of anti-inflammatory drugs,statins, diuretics, beta-blockers, angiotensin converting enzymeinhibitors and angiotensin II receptor blockers. The additionaltherapeutic agent may be aspirin. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more mTAs, wherein the one or moremodified therapeutic agents (mTAs) comprise (a) one or more half-lifeextending moieties, wherein the one or more half-life extending moietiescomprises an extended recombinant polypeptide (XTEN) comprising (i) anamino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; and (b) one or more therapeutic agents (TA),wherein the one or more half-life extending moieties are attached to theone or more cysteine residues in the one or more therapeutic agents. TheXTEN may comprise an amino acid sequence characterized in that the sumof glycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% thetotal amino acid sequence. The XTEN may comprise an amino acid sequenceselected from SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%,85%, 87%, 90%, 93%, 95%, 97%, or 99% or more homologous to an amino acidsequence of SEQ ID NOs: 66-67. The one or more TAs may comprise GLP-1,Exendin-4, exenatide, oxyntomodulin, glucagon, FGF21, or derivativethereof. The GLP-1 may be a human GLP-1. The FGF21 may be a human FGF21.The one or more TAs may further comprise a linker. The metabolic diseaseor condition may be diabetes. The metabolic disease or condition may beglycogen storage disease, phenylketonuria, maple syrup urine disease,glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency,alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency(MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia,Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or NiemannPick disease. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more mTAs, wherein the one or moremodified therapeutic agents (mTAs) comprise (a) one or half-lifeextending moieties, wherein the one or more half-life extending moietiescomprises an extended recombinant polypeptide (XTEN) comprising (i) anamino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; and (b) a therapeutic agent (TA), wherein the TAis a peptide and the one or more half-life extending moieties areattached to the TA via an amino acid residue on the peptide. The XTENmay comprise an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% thetotal amino acid sequence. The XTEN may comprise an amino acid sequenceselected from SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%,85%, 87%, 90%, 93%, 95%, 97%, or 99% or more homologous to an amino acidsequence of SEQ ID NOs: 66-67. The peptide may comprise GLP-1,Exendin-4, exenatide, oxyntomodulin, glucagon, FGF21, or derivativethereof. The GLP-1 may be a human GLP-1. The FGF21 may be a human FGF21.The peptide may further comprise a linker. The metabolic disease orcondition may be diabetes. The metabolic disease or condition may beglycogen storage disease, phenylketonuria, maple syrup urine disease,glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency,alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency(MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia,Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or NiemannPick disease. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more modified therapeutic agents(mTAs) of Formula IIa): TA-A¹-P¹-X, wherein TA is a therapeutic agent;A¹ is a chemical group linking TA and P¹; P¹ is a bond or -PEG-A²-; PEGis a chemical group comprising one or more polyethyleneglycol subunits;A² is a chemical group linking PEG and X; and X is a half-life extendingmoiety. The half-life extending moiety may comprise an extendedrecombinant polypeptide (XTEN) comprising (i) an amino acid sequencecharacterized in that the sum of glycine (G), alanine (A), serine (S),threonine (T), glutamate (E), aspartate (D), leucine (L) and proline (P)residues constitutes more than about 70% of the total amino acidsequence; (ii) a substantially non-repetitive amino acid sequence; (iii)an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets; and P¹ is a bond. The TA may comprise GLP-1, Exendin-4,exenatide, oxyntomodulin, glucagon, FGF21, or derivative thereof. TheGLP-1 may be a human GLP-1. The FGF21 may be a human FGF21. The XTEN maycomprise an amino acid sequence characterized in that the sum of glycine(G), alanine (A), serine (S), threonine (T), glutamate (E), aspartate(D), leucine (L) and proline (P) residues constitutes more than about75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% the total aminoacid sequence. The XTEN may comprise an amino acid sequence selectedfrom SEQ ID NOs: 66-67. The XTEN may comprise an amino acid sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%,90%, 93%, 95%, 97%, or 99% or more homologous to an amino acid sequenceof SEQ ID NOs: 66-67. The metabolic disease or condition may bediabetes. The metabolic disease or condition may be glycogen storagedisease, phenylketonuria, maple syrup urine disease, glutaric acidemiatype 1, Carbamoyl phosphate synthetase I deficiency, alcaptonuria,Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), acuteintermittent porphyria, Lesch-Nyhan syndrome, lipoid congenital adrenalhyperplasia, congenital adrenal hyperplasia, Kearns-Sayre syndrome,Zellweger syndrome, Gaucher's disease, or Niemann Pick disease. The mTAmay be an XTEN-mTA.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more modified therapeutic agents(mTAs) of Formula (I): TA-A¹-P¹-X, wherein TA is a therapeutic agent; A¹is a chemical group linking TA and P¹ or X; P¹ is a bond or comprisespolyglycol; and X is a half-life extending moiety. The half-lifeextending moiety comprises an extended recombinant polypeptide (XTEN)comprising (i) an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 70% of the total amino acid sequence; (ii) a substantiallynon-repetitive amino acid sequence; (iii) an amino acid sequence thathas less than about 10% alpha helices; and/or (iv) an amino acidsequence that has less than about 10% beta-sheets. The XTEN may comprisean amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 75%,77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% the total amino acidsequence. The XTEN may comprise an amino acid sequence selected from SEQID NOs: 66-67. The XTEN may comprise an amino acid sequence that is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%,95%, 97%, or 99% or more homologous to an amino acid sequence of SEQ IDNOs: 66-67. The TA may comprise GLP-1, exendin-4, exenatide,oxyntomodulin, glucagon, FGF21, or derivative thereof. The GLP-1 may bea human GLP-1. The FGF21 may be a human FGF21. The metabolic disease orcondition may be diabetes. The metabolic disease or condition may beglycogen storage disease, phenylketonuria, maple syrup urine disease,glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency,alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency(MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia,Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or NiemannPick disease. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more mTAs, wherein the one or moremodified therapeutic agents (mTAs) comprise (a) one or more half-lifeextending moieties, wherein the one or more half-life extending moietiescomprises an extended recombinant polypeptide (XTEN) comprising (i) anamino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; and (b) one or more therapeutic agents (TAs),wherein the one or more half-life extending moieties are attached to atleast one cysteine residue in the one or more therapeutic agents. TheXTEN may comprise an amino acid sequence characterized in that the sumof glycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% thetotal amino acid sequence. The XTEN may comprise an amino acid sequenceselected from SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%,85%, 87%, 90%, 93%, 95%, 97%, or 99% or more homologous to an amino acidsequence of SEQ ID NOs: 66-67. The one or more TAs may comprise GLP-1,exendin-4, exenatide, oxyntomodulin, glucagon, or derivative thereof.The GLP-1 may be a human GLP-1. The CNS disorder may be Alzheimer'sdisease (AD). Additional CNS disorders include, but are not limited to,encephalitis, meningitis, tropical spastic paraparesis, arachnoid cysts,Huntington's disease, locked-in syndrome, Parkinson's disease,Tourette's, and multiple sclerosis. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more mTAs, wherein the one or moremodified therapeutic agents (mTAs) comprise (a) one or more half-lifeextending moieties, wherein the one or more half-life extending moietiescomprises an extended recombinant polypeptide (XTEN) comprising (i) anamino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 70% ofthe total amino acid sequence; (ii) a substantially non-repetitive aminoacid sequence; (iii) an amino acid sequence that has less than about 10%alpha helices; and/or (iv) an amino acid sequence that has less thanabout 10% beta-sheets; and (b) a therapeutic agent (TA), wherein the TAis a peptide and the one or more half-life extending moieties areattached to the TA via an amino acid residue on the peptide. The XTENmay comprise an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% thetotal amino acid sequence. The XTEN may comprise an amino acid sequenceselected from SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%,85%, 87%, 90%, 93%, 95%, 97%, or 99% or more homologous to an amino acidsequence of SEQ ID NOs: 66-67. The TA may comprise GLP-1, exendin-4,exenatide, oxyntomodulin, glucagon, or derivative thereof. The GLP-1 maybe a human GLP-1. The CNS disorder may be Alzheimer's disease (AD).Additional CNS disorders include, but are not limited to, encephalitis,meningitis, tropical spastic paraparesis, arachnoid cysts, Huntington'sdisease, locked-in syndrome, Parkinson's disease, Tourette's, andmultiple sclerosis. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more modified therapeutic agents(mTAs) of Formula IIa): TA-A¹-P¹-X, wherein TA is a therapeutic agent;A¹ is a chemical group linking TA and P¹; P¹ is a bond or -PEG-A²-; PEGis a chemical group comprising one or more polyethyleneglycol subunits;A² is a chemical group linking PEG and X; and X is a half-life extendingmoiety. The half-life extending moiety may comprise an extendedrecombinant polypeptide (XTEN) comprising (i) an amino acid sequencecharacterized in that the sum of glycine (G), alanine (A), serine (S),threonine (T), glutamate (E), aspartate (D), leucine (L) and proline (P)residues constitutes more than about 70% of the total amino acidsequence; (ii) a substantially non-repetitive amino acid sequence; (iii)an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets. The XTEN may comprise an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%,97%, or 99% the total amino acid sequence; and P¹ is a bond. The XTENmay comprise an amino acid sequence selected from SEQ ID NOs: 66-67. TheXTEN may comprise an amino acid sequence that is at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, or 99%or more homologous to an amino acid sequence of SEQ ID NOs: 66-67. TheTA may comprise GLP-1, exendin-4, exenatide, oxyntomodulin, glucagon, orderivative thereof. The GLP-1 may be a human GLP-1. The CNS disorder maybe Alzheimer's disease (AD). Additional CNS disorders include, but arenot limited to, encephalitis, meningitis, tropical spastic paraparesis,arachnoid cysts, Huntington's disease, locked-in syndrome, Parkinson'sdisease, Tourette's, and multiple sclerosis. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering to the subject one or more modified therapeutic agents(mTAs) of Formula (II): TA-A¹-P¹-X, wherein TA is a therapeutic agent;A¹ is a chemical group linking TA and P¹ or X; P¹ is a bond or comprisespolyglycol; and X is a half-life extending moiety. The half-lifeextending moiety may comprise an extended recombinant polypeptide (XTEN)comprising (i) an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 70% of the total amino acid sequence; (ii) a substantiallynon-repetitive amino acid sequence; (iii) an amino acid sequence thathas less than about 10% alpha helices; and/or (iv) an amino acidsequence that has less than about 10% beta-sheets. The XTEN may comprisean amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 75%,77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% the total amino acidsequence; and P¹ is a bond. The XTEN may comprise an amino acid sequenceselected from SEQ ID NOs: 66-67. The XTEN may comprise an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%,85%, 87%, 90%, 93%, 95%, 97%, or 99% or more homologous to an amino acidsequence of SEQ ID NOs: 66-67. The TA may comprise GLP-1, exendin-4,exenatide, oxyntomodulin, glucagon, or derivative thereof. The GLP-1 maybe a human GLP-1. The CNS disorder may be Alzheimer's disease (AD).Additional CNS disorders include, but are not limited to, encephalitis,meningitis, tropical spastic paraparesis, arachnoid cysts, Huntington'sdisease, locked-in syndrome, Parkinson's disease, Tourette's, andmultiple sclerosis. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more mTAs, wherein the one or more modified therapeuticagents (mTAs) comprise (a) one or more half-life extending moieties,wherein the one or more half-life extending moieties comprises anextended recombinant polypeptide (XTEN) comprising (i) an amino acidsequence characterized in that the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E), aspartate (D), leucine (L) andproline (P) residues constitutes more than about 70% of the total aminoacid sequence; (ii) a substantially non-repetitive amino acid sequence;(iii) an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets; and (b) one or more therapeutic agents (TAs), wherein theone or more half-life extending moieties are attached to at least onecysteine residue in the one or more therapeutic agents. The XTEN maycomprise an amino acid sequence characterized in that the sum of glycine(G), alanine (A), serine (S), threonine (T), glutamate (E), aspartate(D), leucine (L) and proline (P) residues constitutes more than about75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% the total aminoacid sequence. The XTEN may comprise an amino acid sequence selectedfrom SEQ ID NOs: 66-67. The XTEN may comprise an amino acid sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%,90%, 93%, 95%, 97%, or 99% or more homologous to an amino acid sequenceof SEQ ID NOs: 66-67. The one or more TAs may comprise GLP-1, exendin-4,exenatide, oxyntomodulin, glucagon, or derivative thereof. The GLP-1 maybe a human GLP-1. The disease or condition may be a metabolic disease ordisorder. The disease or condition may be diabetes. The disease orcondition may be obesity. Additional diseases and/or conditions whichbenefit from a GLP-1R and/or GCGR agonist include, but are not limitedto, dyslipidemia, cardiovascular and fatty liver diseases. The mTA maybe an XTEN-mTA.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more mTAs, wherein the one or more modified therapeuticagents (mTAs) comprise (a) one or more half-life extending moieties,wherein the one or more half-life extending moieties comprises anextended recombinant polypeptide (XTEN) comprising (i) an amino acidsequence characterized in that the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E), aspartate (D), leucine (L) andproline (P) residues constitutes more than about 70% of the total aminoacid sequence; (ii) a substantially non-repetitive amino acid sequence;(iii) an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets; and (b) a therapeutic agent (TA), wherein the TA is apeptide and the one or more half-life extending moieties are attached tothe TA via an amino acid residue on the peptide. The XTEN may comprisean amino acid sequence characterized in that the sum of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), aspartate (D),leucine (L) and proline (P) residues constitutes more than about 75%,77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% the total amino acidsequence. The XTEN may comprise an amino acid sequence selected from SEQID NOs: 66-67. The XTEN may comprise an amino acid sequence that is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%,95%, 97%, or 99% or more homologous to an amino acid sequence of SEQ IDNOs: 66-67. The TA may comprise GLP-1, exendin-4, exenatide,oxyntomodulin, glucagon, or derivative thereof. The GLP-1 may be a humanGLP-1. The disease or condition may be a metabolic disease or disorder.The disease or condition may be diabetes. The disease or condition maybe obesity. Additional diseases and/or conditions which benefit from aGLP-1R and/or GCGR agonist include, but are not limited to,dyslipidemia, cardiovascular and fatty liver diseases. The mTA may be anXTEN-mTA.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more modified therapeutic agents (mTAs) of Formula IIa):TA-A¹-P¹-X, wherein TA is a therapeutic agent; A¹ is a chemical grouplinking TA and P¹; P¹ is a bond or -PEG-A²-; PEG is a chemical groupcomprising one or more polyethyleneglycol subunits; A² is a chemicalgroup linking PEG and X; and X is a half-life extending moiety. Thehalf-life extending moiety may comprise an extended recombinantpolypeptide (XTEN) comprising (i) an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 70% of the total amino acid sequence; (ii) asubstantially non-repetitive amino acid sequence; (iii) an amino acidsequence that has less than about 10% alpha helices; and/or (iv) anamino acid sequence that has less than about 10% beta-sheets. The XTENmay comprise an amino acid sequence characterized in that the sum ofglycine (G), alanine (A), serine (S), threonine (T), glutamate (E),aspartate (D), leucine (L) and proline (P) residues constitutes morethan about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%, 97%, or 99% thetotal amino acid sequence; and P¹ is a bond. The XTEN may comprise anamino acid sequence selected from SEQ ID NOs: 66-67. The XTEN maycomprise an amino acid sequence that is at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, or 99% or morehomologous to an amino acid sequence of SEQ ID NOs: 66-67. The TA maycomprise GLP-1, exendin-4, exenatide, oxyntomodulin, glucagon, orderivative thereof. The GLP-1 may be a human GLP-1. The disease orcondition may be a metabolic disease or disorder. The disease orcondition may be diabetes. The disease or condition may be obesity.Additional diseases and/or conditions which benefit from a GLP-1R and/orGCGR agonist include, but are not limited to, dyslipidemia,cardiovascular and fatty liver diseases. The mTA may be an XTEN-mTA.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more modified therapeutic agents (mTAs) of Formula (I):TA-A¹-P¹-X, wherein TA is a therapeutic agent; A¹ is a chemical grouplinking TA and P¹ or X; P¹ is a bond or comprises polyglycol; and X is ahalf-life extending moiety. The half-life extending moiety may comprisean extended recombinant polypeptide (XTEN) comprising (i) an amino acidsequence characterized in that the sum of glycine (G), alanine (A),serine (S), threonine (T), glutamate (E), aspartate (D), leucine (L) andproline (P) residues constitutes more than about 70% of the total aminoacid sequence; (ii) a substantially non-repetitive amino acid sequence;(iii) an amino acid sequence that has less than about 10% alpha helices;and/or (iv) an amino acid sequence that has less than about 10%beta-sheets. The XTEN may comprise an amino acid sequence characterizedin that the sum of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E), aspartate (D), leucine (L) and proline (P) residuesconstitutes more than about 75%, 77%, 80%, 83%, 85%, 87%, 90%, 92%, 95%,97%, or 99% the total amino acid sequence. The XTEN may comprise anamino acid sequence selected from SEQ ID NOs: 66-67. The XTEN maycomprise an amino acid sequence that is at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, or 99% or morehomologous to an amino acid sequence of SEQ ID NOs: 66-67. The TA maycomprise GLP-1, exendin-4, exenatide, oxyntomodulin, glucagon, orderivative thereof. The GLP-1 may be a human GLP-1. The disease orcondition may be a metabolic disease or disorder. The disease orcondition may be diabetes. The disease or condition may be obesity.Additional diseases and/or conditions which benefit from a GLP-1R and/orGCGR agonist include, but are not limited to, dyslipidemia,cardiovascular and fatty liver diseases. The mTA may be an XTEN-mTA.

Further disclosed herein are XTEN-mTAs for treating, alleviating,inhibiting and/or preventing one or more diseases and/or conditions. Thedisease and/or condition may be a chronic disease or condition.Alternatively, the disease and/or condition may be an acute disease orcondition. The disease or condition may be recurrent, refractory,accelerated, or in remission. The disease or condition may affect one ormore cell types. The one or more diseases and/or conditions may be anautoimmune disease, inflammatory disease, cardiovascular disease andpregnancy. The XTEN-mTAs disclosed herein may be administered to asubject in need thereof.

Compositions

Disclosed herein are pharmaceutical compositions comprising a modifiedtherapeutic agent disclosed herein. The compositions may comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more modified therapeutic agents. Themodified therapeutic agents may be different. Alternatively, themodified therapeutic agents may be the same or similar. The modifiedtherapeutic agents may comprise different therapeutic agents, differenthalf-life extending moieties, or a combination thereof. The half-lifeextending moieties may be the same or similar.

Further disclosed herein are pharmaceutical compositions comprising anLC disclosed herein. The compositions may comprise 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more LCs. The LCs may be different. Alternatively, the LCsmay be the same or similar. The LCs may comprise different therapeuticagents, different lipids, or a combination thereof. The lipids may bethe same or similar.

The compositions described herein may further comprise one or morepharmaceutically acceptable salts, excipients, or vehicles.Pharmaceutically acceptable salts, excipients, or vehicles for use inthe present pharmaceutical compositions include carriers, excipients,diluents, antioxidants, preservatives, coloring, flavoring and dilutingagents, emulsifying agents, suspending agents, solvents, fillers,bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials, andsurfactants.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. The pharmaceutical compositions may includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers may be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition may be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g., polysorbate 20,polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol)phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodiumlaurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™. series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g., fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions may be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, or intralesional routes. A parenteral formulationtypically may be a sterile, pyrogen-free, isotonic aqueous solution,optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, anti-oxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980.

Pharmaceutical compositions described herein may be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) and/orincreased stability or half-life in a particular local environment. Thecompositions can include the formulation of LCs, polypeptides, nucleicacids, or vectors disclosed herein with particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid, etc., aswell as agents such as a biodegradable matrix, injectable microspheres,microcapsular particles, microcapsules, bioerodible particles beads,liposomes, and implantable delivery devices that provide for thecontrolled or sustained release of the active agent which then can bedelivered as a depot injection. Techniques for formulating suchsustained- or controlled-delivery means are known and a variety ofpolymers have been developed and used for the controlled release anddelivery of drugs. Such polymers are typically biodegradable andbiocompatible. Polymer hydrogels, including those formed by complexationof enantiomeric polymer or polypeptide segments, and hydrogels withtemperature or pH sensitive properties, may be desirable for providingdrug depot effect because of the mild and aqueous conditions involved intrapping bioactive protein agents (e.g., antibodies comprising anultralong CDR3). See, for example, the description of controlled releaseporous polymeric microparticles for the delivery of pharmaceuticalcompositions in WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al.,Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al.,Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present disclosure. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humor of the eye. Esterified derivatives of hyaluronic acid havebeen used to produce microspheres for use in delivery that arebiocompatible and biodegradable (see, for example, Cortivo et al.,Biomaterials (1991) 12:727-730; EP 517,565; WO 96/29998; Illum et al.,J. Controlled Rel. (1994) 29:133-141).

Both biodegradable and non-biodegradable polymeric matrices may be usedto deliver compositions of the present disclosure, and such polymericmatrices may comprise natural or synthetic polymers. Biodegradablematrices are preferred. The period of time over which release occurs isbased on selection of the polymer. Typically, release over a periodranging from between a few hours and three to twelve months is mostdesirable. Exemplary synthetic polymers which may be used to form thebiodegradable delivery system include: polymers of lactic acid andglycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, polyalkylene terepthalates, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides,polyurethanes and co-polymers thereof, poly(butic acid), poly(valericacid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, polymers of acrylic and methacrylicesters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxylethyl cellulose, cellulose triacetate,cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene andpolyvinylpyrrolidone. Exemplary natural polymers include alginate andother polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion. The polymer optionally is in the formof a hydrogel (see, for example, WO 04/009664, WO 05/087201, Sawhney, etal., Macromolecules, 1993, 26, 581-587) that can absorb up to about 90%of its weight in water and further, optionally is cross-linked withmulti-valent ions or other polymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di- and triglycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; JP 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324).

Alternatively or additionally, the compositions may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which an LC, nucleic acid encodingat least a portion of an LC, or vector comprising a nucleic acidencoding at least a portion of an LC disclosed herein has been absorbedor encapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of an LC,nucleic acid encoding at least a portion of an LC, or vector comprisinga nucleic acid encoding at least a portion of an LC disclosed herein canbe directly through the device via bolus, or via continuousadministration, or via catheter using continuous infusion.

A pharmaceutical composition comprising an LC, nucleic acid encoding atleast a portion of an LC, or vector comprising a nucleic acid encodingat least a portion of an LC disclosed herein may be formulated forinhalation, such as for example, as a dry powder. Inhalation solutionsalso may be formulated in a liquefied propellant for aerosol delivery.In yet another formulation, solutions may be nebulized. Additionalpharmaceutical composition for pulmonary administration include, thosedescribed, for example, in WO 94/20069, which discloses pulmonarydelivery of chemically modified proteins. For pulmonary delivery, theparticle size should be suitable for delivery to the distal lung. Forexample, the particle size may be from 1 μm to 5 μm; however, largerparticles may be used, for example, if each particle is fairly porous.

Certain formulations comprising an LC, nucleic acid encoding at least aportion of an LC, or vector comprising a nucleic acid encoding at leasta portion of an LC disclosed herein may be administered orally.Formulations administered in this fashion may be formulated with orwithout those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents may be includedto facilitate absorption of a selective binding agent. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders also can beemployed.

Another preparation may involve an effective quantity of an antibodycomprising an LC, nucleic acid encoding at least a portion of an LC, orvector comprising a nucleic acid encoding at least a portion of an LCdisclosed herein in a mixture with non-toxic excipients which aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or another appropriate vehicle, solutions may be preparedin unit dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations may be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose may be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages may be ascertained through use of appropriatedose-response data.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatis pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” refers toan excipient, carrier or adjuvant that may be administered to a subject,together with at least one antibody of the present disclosure, and whichdoes not destroy the pharmacological activity thereof and is nontoxicwhen administered in doses sufficient to deliver a therapeutic amount ofthe compound.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient, or carrier with which at least one antibody of the presentdisclosure is administered.

Vectors, Host Cells and Recombinant Methods

A TA, as disclosed herein, may be expressed by recombinant methods.Generally, a nucleic acid encoding a TA may be isolated and insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. DNA encoding the LC may be prepared by PCRamplification and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically tonucleotides encoding LC). In an exemplary embodiment, a nucleic acidencoding an LC is PCR amplified, restriction enzyme digested and gelpurified. The digested LC may be inserted into a replicable vector. Thereplicable vector containing the digested LC insertion may betransformed or transduced into a host cell for further cloning(amplification of the DNA) or for expression. Host cells may beprokaryotic or eukaryotic cells.

Polynucleotide sequences encoding polypeptide components of the LCsdisclosed herein may be obtained by PCR amplification with overlappingoligonucleotide primers. Polynucleotide sequences may be isolated andsequenced from TA producing cells. Alternatively, polynucleotides may besynthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in prokaryotic and/or eukaryotic hosts.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism may be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

TAs may be expressed intracellularly (e.g., cytoplasm) orextracellularly (e.g., secretion). For extracellular expression, thevector may comprise a secretion signal which enables translocation ofthe TA to the outside of the cell.

Suitable host cells for cloning or expression of TA-encoding vectorsinclude prokaryotic or eukaryotic cells. The host cell may be aeukaryotic. Examples of eukaryotic cells include, but are not limitedto, Human Embryonic Kidney (HEK) cell, Chinese Hamster Ovary (CHO) cell,fungi, yeasts, invertebrate cells (e.g., plant cells and insect cells),lymphoid cell (e.g., YO, NSO, Sp20 cell). Other examples of suitablemammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7); baby hamster kidney cells (BHK); mouse Sertoli cells; monkeykidney cells (CV 1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells(HepG2); mouse mammary tumor (MMT 060562); TR1 cells; MRC 5 cells; andFS4 cells. The host cell may be a prokaryotic cell (e.g., E. coli).

Host cells may be transformed with vectors containing nucleotidesencoding a TA. Transformed host cells may be cultured in media. Themedia may be supplemented with one or more agents for inducingpromoters, selecting transformants, or amplifying or expressing thegenes encoding the desired sequences. Methods for transforming hostcells are known in the art and may include electroporation, calciumchloride, or polyethylene glycol/DMSO.

Alternatively, host cells may be transfected or transduced with vectorscontaining nucleotides encoding a TA. Transfected or transduced hostcells may be cultured in media. The media may be supplemented with oneor more agents for inducing promoters, selecting transfected ortransduced cells, or expressing genes encoding the desired sequences.

The expressed TAs may be secreted into and recovered from the periplasmof the host cells or transported into the culture media. Proteinrecovery from the periplasm may involve disrupting the host cell.Disruption of the host cell may comprise osmotic shock, sonication orlysis. Centrifugation or filtration may be used to remove cell debris orwhole cells. The TAs may be further purified, for example, by affinityresin chromatography.

Alternatively, TAs that are secreted into the culture media may beisolated therein. Cells may be removed from the culture and the culturesupernatant being filtered and concentrated for further purification ofthe proteins produced. The expressed polypeptides can be furtherisolated and identified using commonly known methods such aspolyacrylamide gel electrophoresis (PAGE) and Western blot assay.

TA production may be conducted in large quantity by a fermentationprocess. Various large-scale fed-batch fermentation procedures areavailable for production of recombinant proteins. Large-scalefermentations have at least 1000 liters of capacity, preferably about1,000 to 100,000 liters of capacity. These fermentors may use agitatorimpellers to distribute oxygen and nutrients, especially glucose (apreferred carbon/energy source). Small scale fermentation refersgenerally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described herein. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction times may be used.

To improve the production yield and quality of the TAs disclosed herein,various fermentation conditions can be modified. For example, to improvethe proper assembly and folding of the secreted TA polypeptides,additional vectors overexpressing chaperone proteins, such as Dsbproteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolylcis, trans-isomerase with chaperone activity) may be used toco-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the presentdisclosure. For example, host cell strains may be modified to effectgenetic mutation(s) in the genes encoding known bacterial proteases suchas Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available.

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography and gelfiltration using, for example, Sephadex G-75.

TAs may be concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pellicon®ultrafiltration unit.

Protease inhibitors or protease inhibitor cocktails may be included inany of the foregoing steps to inhibit proteolysis of the TA.

In some cases, a TA or fragment thereof may not be biologically activeupon isolation. Various methods for “refolding” or converting apolypeptide to its tertiary structure and generating disulfide linkages,can be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. The refolding/oxidation solution may also contain areducing agent or the reducing agent plus its oxidized form in aspecific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridge(s). Some of the commonly used redox couples includecystein/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-mercaptoethanol(bME)/di-thio-b(ME). In many instances, a cosolvent maybe used to increase the efficiency of the refolding, and common reagentsused for this purpose include glycerol, polyethylene glycol of variousmolecular weights, arginine and the like. Choice of buffer may determinethe three-dimensional structure of the refolded peptide. Use of a buffercomprising ammonium sulfate may result in the desired refolded peptidein better yield than other types of buffers.

Kits/Articles of Manufacture

As an additional aspect, the present disclosure includes kits whichcomprise one or more compounds or compositions packaged in a mannerwhich facilitates their use to practice methods of the presentdisclosure. In one embodiment, such a kit includes a compound orcomposition described herein (e.g. a mTA alone or in combination with asecond agent), packaged in a container with a label affixed to thecontainer or a package insert that describes use of the compound orcomposition in practicing the method. Suitable containers include, forexample, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The container mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises a mTA as disclosed herein; and (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises a further therapeutic agent. The article of manufacture inthis embodiment disclosed herein may further comprise a package insertindicating that the first and second compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes. Preferably, the compound orcomposition is packaged in a unit dosage form. The kit may furtherinclude a device suitable for administering the composition according toa specific route of administration or for practicing a screening assay.Preferably, the kit contains a label that describes use of the modifiedtherapeutic agent composition.

In certain embodiments, the composition comprising the modifiedtherapeutic agent is formulated in accordance with routine procedures asa pharmaceutical composition adapted for intravenous administration tomammals, such as humans, bovines, felines, canines, and murines.Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilising agent and a local anaesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientsmay be mixed prior to administration.

The amount of the composition described herein which may be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a protein can bedetermined by standard clinical techniques. In addition, in vitro assaysmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation may also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses are extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

“Amino” refers to the —NH₂ radical.

“Cyano” or “nitrile” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Oxime” refers to the ═N—OH substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radical, hasfrom one to thirty carbon atoms, and is attached to the rest of themolecule by a single bond. Alkyls comprising any number of carbon atomsfrom 1 to 30 are included. An alkyl comprising up to 30 carbon atoms isreferred to as a C₁-C₃₀ alkyl, likewise, for example, an alkylcomprising up to 12 carbon atoms is a C₁-C₁₂ alkyl. Alkyls (and othermoieties defined herein) comprising other numbers of carbon atoms arerepresented similarly. Alkyl groups include, but are not limited to,C₁-C₃₀ alkyl, C₁-C₂₀ alkyl, C₁-C₁₅ alkyl, C₁-C₁₀ alkyl, C₁-C₈ alkyl,C₁-C₆ alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl, C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈alkyl and C₄-C₈ alkyl. Representative alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl),3-methylhexyl, 2-methylhexyl, vinyl, allyl, propynyl, and the like.Alkyl comprising unsaturations include alkenyl and alkynyl groups.Unless stated otherwise specifically in the specification, an alkylgroup may be optionally substituted as described below.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain, as described for alkyl above. Unless stated otherwisespecifically in the specification, an alkylene group may be optionallysubstituted as described below.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined. Unless stated otherwise specifically in thespecification, an alkoxy group may be optionally substituted asdescribed below.

“Aryl” refers to a radical derived from a hydrocarbon ring systemcomprising hydrogen, 6 to 30 carbon atoms and at least one aromaticring. The aryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryl radicals include, but are not limited to, aryl radicalsderived from the hydrocarbon ring systems of aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane,indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, andtriphenylene. Unless stated otherwise specifically in the specification,the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant toinclude aryl radicals that are optionally substituted.

“Cycloalkyl” or “carbocycle” refers to a stable, non-aromatic,monocyclic or polycyclic carbocyclic ring, which may include fused orbridged ring systems, which is saturated or unsaturated. Representativecycloalkyls or carbocycles include, but are not limited to, cycloalkylshaving from three to fifteen carbon atoms, from three to ten carbonatoms, from three to eight carbon atoms, from three to six carbon atoms,from three to five carbon atoms, or three to four carbon atoms.Monocyclic cycloalkyls or carbocycles include, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Polycyclic cycloalkyls or carbocycles include, for example, adamantyl,norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane,cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane,and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless otherwise statedspecifically in the specification, a cycloalkyl or carbocycle group maybe optionally substituted. Illustrative examples of cycloalkyl groupsinclude, but are not limited to, the following moieties:

and the like.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure. When the fused ring is a heterocyclyl ringor a heteroaryl ring, any carbon atom on the existing ring structurewhich becomes part of the fused heterocyclyl ring or the fusedheteroaryl ring may be replaced with a nitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl,2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl,1,2-dibromoethyl, and the like. Unless stated otherwise specifically inthe specification, a haloalkyl group may be optionally substituted.

“Haloalkoxy” similarly refers to a radical of the formula —OR_(a) whereR_(a) is a haloalkyl radical as defined. Unless stated otherwisespecifically in the specification, a haloalkoxy group may be optionallysubstituted as described below.

“Heterocycloalkyl” or “heterocyclyl” or “heterocyclic ring” or“heterocycle” refers to a stable 3- to 24-membered non-aromatic ringradical comprising 2 to 23 carbon atoms and from one to 8 heteroatomsselected from the group consisting of nitrogen, oxygen, phosphorous andsulfur. Unless stated otherwise specifically in the specification, theheterocyclyl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclyl radical may be partially or fullysaturated. Examples of such heterocyclyl radicals include, but are notlimited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl,tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, 12-crown-4,15-crown-5, 18-crown-6, 21-crown-7, aza-18-crown-6, diaza-18-crown-6,aza-21-crown-7, and diaza-21-crown-7. Unless stated otherwisespecifically in the specification, a heterocyclyl group may beoptionally substituted. Illustrative examples of heterocycloalkylgroups, also referred to as non-aromatic heterocycles, include:

and the like. The term heterocycloalkyl also includes all ring forms ofthe carbohydrates, including but not limited to the monosaccharides, thedisaccharides and the oligosaccharides. Unless otherwise noted,heterocycloalkyls have from 2 to 10 carbons in the ring. It isunderstood that when referring to the number of carbon atoms in aheterocycloalkyl, the number of carbon atoms in the heterocycloalkyl isnot the same as the total number of atoms (including the heteroatoms)that make up the heterocycloalkyl (i.e. skeletal atoms of theheterocycloalkyl ring). Unless stated otherwise specifically in thespecification, a heterocycloalkyl group may be optionally substituted.

The term “heteroaryl” as used herein, alone or in combination, refers tooptionally substituted aromatic monoradicals containing from about fiveto about twenty skeletal ring atoms, where one or more of the ring atomsis a heteroatom independently selected from among oxygen, nitrogen,sulfur, phosphorous, silicon, selenium and tin but not limited to theseatoms and with the proviso that the ring of said group does not containtwo adjacent O or S atoms. In embodiments in which two or moreheteroatoms are present in the ring, the two or more heteroatoms can bethe same as each another, or some or all of the two or more heteroatomscan each be different from the others. The term heteroaryl includesoptionally substituted fused and non-fused heteroaryl radicals having atleast one heteroatom. The term heteroaryl also includes fused andnon-fused heteroaryls having from five to about twelve skeletal ringatoms, as well as those having from five to about ten skeletal ringatoms. Bonding to a heteroaryl group can be via a carbon atom or aheteroatom. Thus, as a non-limiting example, an imidiazole group may beattached to a parent molecule via any of its carbon atoms(imidazol-2-yl, imidazol-4-yl or imidazol-5-yl), or its nitrogen atoms(imidazol-1-yl or imidazol-3-yl). Likewise, a heteroaryl group may befurther substituted via any or all of its carbon atoms, and/or any orall of its heteroatoms. A fused heteroaryl radical may contain from twoto four fused rings where the ring of attachment is a heteroaromaticring and the other individual rings may be alicyclic, heterocyclic,aromatic, heteroaromatic or any combination thereof. A non-limitingexample of a single ring heteroaryl group includes pyridyl; fused ringheteroaryl groups include benzimidazolyl, quinolinyl, acridinyl; and anon-fused bi-heteroaryl group includes bipyridinyl. Further examples ofheteroaryls include, without limitation, furanyl, thienyl, oxazolyl,acridinyl, azepinyl, phenazinyl, benzimidazolyl, benzindolyl,benzofuranyl, benzofuranonyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, benzothiophenyl, benzoxadiazolyl, benzodioxolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzotriazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzothienyl (benzothiophenyl),benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,dibenzofuranyl, dibenzothiophenyl, furanonyl, imidazolyl, indolyl,isoxazolyl, isoquinolinyl, indolizinyl, indazolyl, isoindolyl,indolinyl, isoindolinyl, indolizinyl, isothiazolyl,isoindolyloxadiazolyl, indazolyl, naphthyridinyl, oxadiazolyl,2-oxoazepinyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl,1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl,phenothiazinyl, phenoxazinyl, pyridinyl, pyridazinyl, pyrimidinyl,pyrazinyl, pyrrolyl, pyrazinyl, pyrazolyl, purinyl, phthalazinyl,pteridinyl, quinolinyl, quinazolinyl, quinoxalinyl, quinuclidinyl,triazolyl, tetrazolyl, thiazolyl, triazinyl, thiadiazolyl,tetrahydroquinolinyl, thiazolyl, and thiophenyl and the like, and theiroxides, such as for example pyridyl-N-oxide. Illustrative examples ofheteroaryl groups include the following moieties:

and the like.

All the above groups may be either substituted or unsubstituted. Theterm “substituted” as used herein means any of the above groups (e.g.,alkyl, alkylene, alkoxy, aryl, cycloalkyl, haloalkyl, heterocyclyland/or heteroaryl) may be further functionalized wherein at least onehydrogen atom is replaced by a bond to a non-hydrogen atom substituent.Unless stated specifically in the specification, a substituted group mayinclude one or more substituents selected from: oxo, amino, —CO₂H,nitrile, nitro, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, aryl,cycloalkyl, heterocyclyl, heteroaryl, dialkylamines, arylamines,alkylarylamines, diarylamines, trialkylammonium (—N⁺R₃), N-oxides,imides, and enamines; a silicon atom in groups such as trialkylsilylgroups, dialkylarylsilyl groups, alkyldiarylsilyl groups, triarylsilylgroups, perfluoroalkyl or perfluoroalkoxy, for example, trifluoromethylor trifluoromethoxy. “Substituted” also means any of the above groups inwhich one or more hydrogen atoms are replaced by a higher-order bond(e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo,carbonyl, carboxyl, and ester groups; and nitrogen in groups such asimines, oximes, hydrazones, and nitriles. For example, “substituted”includes any of the above groups in which one or more hydrogen atoms arereplaced with —NH₂, —NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h),—NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR₂, —SO₂R_(g),—OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h). In theforegoing, R_(g) and R_(h) are the same or different and independentlyhydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. Inaddition, each of the foregoing substituents may also be optionallysubstituted with one or more of the above substituents. Furthermore, anyof the above groups may be substituted to include one or more internaloxygen, sulfur, or nitrogen atoms. For example, an alkyl group may besubstituted with one or more internal oxygen atoms to form an ether orpolyether group. Similarly, an alkyl group may be substituted with oneor more internal sulfur atoms to form a thioether, disulfide, etc.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl” as defined above.Further, an optionally substituted group may be un-substituted (e.g.,—CH₂CH₃), fully substituted (e.g., —CF₂CF₃), mono-substituted (e.g.,—CH₂CH₂F) or substituted at a level anywhere in-between fullysubstituted and mono-substituted (e.g., —CH₂CHF₂, —CH₂CF₃, —CF₂CH₃,—CFHCHF₂, etc). It will be understood by those skilled in the art withrespect to any group containing one or more substituents that suchgroups are not intended to introduce any substitution or substitutionpatterns (e.g., substituted alkyl includes optionally substitutedcycloalkyl groups, which in turn are defined as including optionallysubstituted alkyl groups, potentially ad infinitum) that are stericallyimpractical and/or synthetically non-feasible. Thus, any substituentsdescribed should generally be understood as having a maximum molecularweight of about 1,000 daltons, and more typically, up to about 500daltons.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The compounds presented herein mayexist as tautomers. Tautomers are compounds that are interconvertible bymigration of a hydrogen atom, accompanied by a switch of a single bondand adjacent double bond. In bonding arrangements where tautomerizationis possible, a chemical equilibrium of the tautomers will exist. Alltautomeric forms of the compounds disclosed herein are contemplated. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Some examples of tautomericinterconversions include:

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes, such as, oxidation reactions) by which aparticular substance is changed by an organism. Thus, enzymes mayproduce specific structural alterations to a compound. For example,cytochrome P450 catalyzes a variety of oxidative and reductive reactionswhile uridine diphosphate glucuronyl transferases catalyze the transferof an activated glucuronic-acid molecule to aromatic alcohols, aliphaticalcohols, carboxylic acids, amines and free sulfhydryl groups. Furtherinformation on metabolism may be obtained from The Pharmacological Basisof Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of thecompounds disclosed herein can be identified either by administration ofcompounds to a host and analysis of tissue samples from the host, or byincubation of compounds with hepatic cells in vitro and analysis of theresulting compounds. Both methods are well known in the art. Metabolitesof a compound may be formed by oxidative processes and correspond to thecorresponding hydroxy-containing compound. A compound may be metabolizedto one or more pharmacologically active metabolites.

As used herein, a “derivative” of a peptide refers to, but is notlimited to, a modified peptide that allows for lipid attachment (such asone or more amino acid residue replacements or L- vs D-amino acidreplacements), a fragment, an analog with one or more additional aminoacids, a complex and/or an aggregate of the peptide. A derivative of apeptide may be a homolog that has at least 50% homology with respect tothe peptide. A derivative of a peptide may be a homolog that has atleast 60% homology with respect to the peptide. A derivative of apeptide may be a homolog that has at least 70% homology with respect tothe peptide. A derivative of a peptide may be a homolog that has atleast 80% homology with respect to the peptide. A derivative of apeptide may be a homolog that has at least 90% homology with respect tothe peptide.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” may refer to: 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder; and/or 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that functionsimilarly to the naturally occurring amino acids. Naturally occurringamino acids are those encoded by the genetic code, as well as thoseamino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

As used herein, the term “therapeutic agent” or “peptide therapeuticagent” refers to a protein or peptide that modulates the activity ofanother protein, peptide, cell or tissue. Modulating the activity cancomprise increasing, decreasing, stimulating, or preventing the activityor expression of the protein, peptide, cell or tissue. Therapeuticagents may modulate the activity of proteins or peptides involved in theetiology of a disease or disorder. Exemplary TAs may include, but arenot limited to, at least a portion of a hormone, kinase, receptor,ligand, growth factor, regulatory protein, metabolic protein, cytokine,chemokine, interferon, phosphatase, antibody or any combination thereof.

“Disorder” or “disease” refers to a condition that would benefit fromtreatment with a substance/molecule (e.g., a mTA as disclosed herein) ormethod disclosed herein. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question.

“Treatment” refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, rodents (e.g., mice and rats), and monkeys;domestic and farm animals; and zoo, sports, laboratory, or pet animals,such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Insome embodiments, the mammal is selected from a human, rodent, ormonkey.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatis pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” refers toan excipient, carrier or adjuvant that can be administered to a subject,together with at least one antibody of the present disclosure, and whichdoes not destroy the pharmacological activity thereof and is nontoxicwhen administered in doses sufficient to deliver a therapeutic amount ofthe compound.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient, or carrier with which at least one antibody of the presentdisclosure is administered.

The terms “modified therapeutic agent,” “mTA,” “lipid conjugate,” and“LC” may be used interchangeably. These terms may refer to a therapeuticagent (TA) attached to a half-life extending moiety.

EXAMPLES Example 1. Construction and Expression of Recombinant Relaxin 2with Linker Between Chain B and Chain a as a CBD Fusion with MutationS26C and S29C

The purpose of this experiment was to construct and express a GGGRGGR(SEQ ID NO: 68) linker between chain B and chain A recombinant relaxin 2with mutation S26C and S29C.

Materials

Materials used for this experiment include:

pVB008 relaxin 2 linker linear

pVB008 relaxin 2 linker linear S26C

pVB008 relaxin 2 linker linear S29C

Oligo stock solution 100 μM of SEQ ID NOs: 38-46 as disclosed in Table6.

FIG. 4 depicts a schematic of pVB008 relaxin2 linker vector.

Step 1. Assembling PCR—Amplification of Relaxin Fragments

PCR reactions to generate relaxin fragments wild type or with S26C orS29C mutations were prepared as follows:

Reaction 1 Reaction 2 Reaction 3 Reaction 4 Reagent (μl) (μl) (μl) (μl)Oligo mix 1 2 4 8 5× Buffer 20 20 20 20 10 mM dNTP 4 4 4 4 One Taq 1 1 11 H₂O 74 73 61 67

Step 2. PCR Program

Relaxin2 fragments (wild type, S26C and S29C) were amplified byconducting the following PCR program:

94° C.-2 Min, followed by 20 cycles of 94° C.-15 Sec, 42° C.-30 Sec, and68° C.-15 Sec

PCR products of the relaxin-2 fragments were run on a 0.8% agarose gel.As shown in FIG. 5, Lanes 1, 6 and 11 contain the 100 bp ladder, Lane2-5 contain the S26C oligo mix Step 1 Reaction 1-4 reactions,respectively, Lane 7-10 contain the S29C oligo mix Step 1 Reaction 1-4reactions, respectively.

Step 3. Amplification of Relaxin2 Linker

PCR reactions to generate a relaxin2 linker were prepared as follows:

Reagent Reaction 1 (μl) Reaction 2 (μl) Reaction 3 (μl) Assembling mix 12 4 5× Buffer 20 20 20 10 mM dNTP 4 4 4 10 μM Primer mix 4 4 4 One Taq 11 1 H₂O 70 69 67

The 10 μM Primer mix contained a Relaxin2 forward primer (pVB008Relaxin2 Amp for (#60): AATCTGTATTTCCAGGGATCCGGTGGTGA=SEQ ID NO: 64 anda Relaxin2 reverse primer (Relaxin2 Linker Amp rev (#211)TGGCTAAGCTTTAGCAGAAACGAGCCAGAGAACGTTTGGTGCAACCAACGTGGC=SEQ ID NO: 65).The PCR amplified reaction 1 mix from step 2 was used for the assemblingmix.

Relaxin 2 linker was amplified by conducting the following PCR program:94° C.-2 Min, followed by 25 cycles of 94° C.-15 Sec, 52° C.-15 Sec, and68° C.-60 Sec.

PCR products of the relaxin-2 linker amplification were run on a 0.8%agarose gel. As shown in FIG. 6, Lane 1 contains the 100 ladder, Lane 2contains S29C Step 3 Reaction 1, Lane 3 contains S29C Step 3 Reaction 2,and Lane 4 contains S29C Step 3 Reaction 3.

Step 4. Digestion of Vector pVB008 and pAC145

A restriction digest of vector pVB008 and pAC145 was performed toprepare the vectors for insertion of the Relaxin 2 linker PCR product.The restriction digest was prepared as follows:

20 μl pVB008/pAC045

10 μl 10× Buffer #4

2 μl HindIII-HF

2 μl BamHI-HF

66 μl H₂O

The restriction digest reaction was incubated at 37° C. for 1 hour.

Step 5. Digestion of Insert

A restriction digest of the Relaxin 2 linker PCR products (wild type,S26C, and S29C) was performed to prepare the PCR product for insertioninto pVB008 and pAC145 vectors. The restriction digest was prepared asfollows:

20 μl full length fragment

10 μl 10× Buffer #4

2 μl HindIII-HF

2 μl BamHI-HF

66 μl H₂O

The restriction digest reaction was incubated at 37° C. for 1 hour.

Step 6. Gel Purification

The pVB008 digested vector, pAC145 digested vector and Relaxin2 linkerinsert were run on an agarose gel. As shown in FIG. 7, Lane 1 containsthe 1 kb ladder, Lane 2 contains the digested pVB008 vector, Lane 3contains the digested pAC145 vector, Lane 4 contains the 100 bp ladder,Lane 5 contains the Relaxin2-linker insert, Lane 6 contains the Relaxin2S26C-linker insert and Lane 7 contains the Relaxin2 S29C-linker insert.The digested vectors and Relaxin2 PCR products were gel purified as perthe manufacturer's instructions (NucleoSpin Gel and PCR CleanupMacherey-Nagel).

Step 7. Ligation/Transformation

The gel-purified digested vector (vector dg) and the digested PCRproduct (relaxin-2 linear dg (Insert)) were ligated by the followingligation reaction:

5 μl Vector dg

1 μl relaxin-2 linear dg (Insert)

2 μl 10× Buffer

1 μl T4 DNA Ligase

11 μl H₂O

The ligation reaction was incubated at room temperature (RT) for 1 hour.

The ligated product was then used to transform NEB turbo Competentcells, 2 μl of the ligation reaction was transformed into NEB turboCompetent Cells according to protocol. The transformed cells were platedon LB/Kan plates.

5 colonies were picked and inoculated in 5 ml LB+Kan. The cells weregrown for 6 hours at 37° C.

Step 8. Preparation of DNA for Sequencing

DNA was prepared from 1.5 ml of each of the 5 cultures using aNucleoSpin DNA Mini Prep Macherey-Nagel according to the protocol.Sequencing of the DNA was performed to verify the insertion of Relaxin2into the pVB008 and pAC145 vector.

Step 9. Expression and Purification of Relaxin Peptides

Relaxin or modified relaxin peptides were expressed through transienttransfections of free style HEK293 cells with nucleotide vectorsencoding such peptides. Expressed peptides were secreted into theculture medium and harvested at 48 and 96 hours after transfection. Thefusion proteins were purified by Protein A/G chromatography (ThermoFisher Scientific, IL), and analyzed by SDS-PAGE gel. FIG. 8 shows anSDS-PAGE gel of expression of relaxin and relaxin mutants. As shown inFIG. 8, Lanes 1 and 17 show protein standards; Lanes 2-6 show pCAC0095lysate, Wash 1, Wash 2, Wash 4 and Inclusion Body, respectively; Lanes7-11 show pCAC0096 lysate, Wash 1, Wash 2, Wash 4 and Inclusion Body,respectively; and Lanes 12-16 show pCAC0097 lysate, Wash 1, Wash 2, Wash4 and Inclusion Body, respectively. PCAC0095 represents wild typerelaxin with GGGRGG (SEQ ID NO: 69) linker and N-terminus 8×His (SEQ IDNO: 70). PCAC0096 represents S26C relaxin with GGGRGG (SEQ ID NO: 69)linker and N-terminus 8×His (SEQ ID NO: 70). PCAC0097 represents S29Crelaxin with GGGRGG (SEQ ID NO: 69) linker and N-terminus 8×His (SEQ IDNO: 70). As shown in FIG. 9, Lanes 1 and 6 show protein standards; Lanes2-5 show pCAC002 Load, Flowthrough, Wash and Beads, respectively.PCAC002 represents wild type relaxin with CBD tag at the N-terminus.

Example 2. Measuring Bioactivity of Relaxin Peptides

Relaxin 2 signals through leucine-rich repeat-containing GPCRs (i.e.LGR7 (RFXR1)). The bioactivity of wild-type relaxin peptides wasdetermined based on the stimulation of adenylate cyclase activity inHEK293T cells stably expressing recombinant LGR7. Stable LGR7 expressingcells were maintained in Dulbecco's modified Eagle's medium/F-12 mediasupplemented with 200 ng/ml Zeocin (Invitrogen). A cyclic AMP responsiveelement driven luciferase (CRE-Luc) reporter line was generated bylentiviral transduction of these LGR7 expressing HEK293T cells withCRE-Luc lentivirus (Qiagen) and selected with puromycine (2 μg/mL) fortwo weeks. A reporter gene assay was used to detect cAMP levels and LGR7activation by relaxin.

Assay detail: HEK293T cells expressing LGR7 and CRE-Luc are seeded at adensity of 5×10³ cells per well in 50 μl of Dulbecco's modified Eagle'smedium/F-12 medium containing 10% FBS in 384-well solid bottom whiteplates. Cells are pre-incubated at 37° C. for overnight. Differentconcentrations of relaxin peptides (from 0.01 nM to 1000 nM) were addedin triplicate, incubated for 18 hours and luciferase activity wasdetected by adding 104 of Bright Glo (Promega). Luminescence wasrecorded on Envision (Perkin Elmer). EC₅₀ is calculated after non-linearcurve fitting (see FIG. 3 for relaxin H2 dose response curve). Selectdata is shown in Tables 2 and 3.

Example 3. Synthesis of Pegylated Lipid (Amide Linkage) ConjugatedRelaxin (Scheme A)

tert-Butyl (2-(2-(2-tetradecanamidoethoxy)ethoxy)ethyl)carbamate (A-2-a)

N-t-Boc-amido-dPEG3-amine (0.5 g, 2.0 mmol) was added to a solution ofmyristic acid (0.46 g, 2.0 mmol) in 10 ml of dry DMF, followed by HATU(0.8 g, 2.1 mmol) and DIEA (0.45 mL, 2.4 mmol). The mixture was stirredat RT for 6 h and the solvent was evaporated in vacuo. The crudematerial was dissolved in EtOAc, washed with cold 1% HCl, saturatedNaHCO₃ and brine, dried over Na₂SO₄, filtered and concentrated. Thecrude material was purified by flash column chromatography on silica gelwith a gradient 25-50% EtOAc in hexanes to afford 0.83 g of desiredcompound as white solids (Yield 90%). m/z (ESI+) 459.6 (M+H).

tert-Butyl(2-(2-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenamido)ethoxy)ethoxy)ethyl)carbamate(A-2-b)

The title compound was prepared using analogous conditions as theprocedure for A-2-a, using N-t-Boc-amido-dPEG3-amine (0.25 g, 1.0 mmol),docosahexaenoic acid (0.33 g, 1.0 mmol), HATU (0.41 g, 1.1 mmol) andDIEA (0.22 mL, 1.2 mmol in dry DMF (5 mL). Yield 68%, brown oil. ¹H NMR(500 MHz; CDCl₃): δ 0.97 (t, J=6.0 Hz, 3H), 1.44 (s, 9H), 2.07 (t, J=5.0Hz, 2H), 2.25 (J=5.0 Hz, 2H), 2.41 (dd, J=5.0, 6.5 Hz, 2H), 2.79-2.87(m, 10H), 3.32 (t, J=5.5 Hz, 2H), 3.45 (dd, J=5.0, 6.0 Hz, 2H), 3.55 (t,J=5.0 Hz, 4H), 3.59-3.62 (m, 4H), 4.97 (s, 1H), 5.30-5.42 (m, 12H), 6.03(s, 1H); m/z (ESI+) 459.6 (M+H).

tert-Butyl(2-(2-(2-((R)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)ethoxy)ethoxy)ethyl)carbamate (A-2-c)

N-t-Boc-amido-dPEG3-amine (0.14 g, 0.55 mmol) was added to a solution ofNHS-activated lithocholic acid ester (0.24 g, 0.5 mmol) in 5 ml of dryDMF, followed by DIEA (0.18 mL, 1.0 mmol). The mixture was stirred at RTfor 16 h, and the solvent was evaporated in vacuo. The crude material isdissolved in DCM, washed with cold 1% HCl, saturated NaHCO₃ and brine,dried over Na₂SO₄, filtered and concentrated. The crude material waspurified by flash column chromatography on silica gel with a gradient1-5% methanol in DCM to afford 0.48 g of desired compound as a whitesolid (Yield 80%). ¹H NMR (500 MHz; CDCl₃): δ 0.62 (s, 3H), 0.90-2.25(m, 30H), 3.31 (s, 2H), 3.43-3.46 (s, 2H), 3.52-3.56 (m, 4H), 3.59-3.60(m, 6H), 4.94 (s, 1H), 6.05 (s, 1H); m/z (ESI+) 628.6 (M+H) m/z (ESI+)607.5 (M+H).

N-(2-(2-(2-(2-Bromoacetamido)ethoxy)ethoxy)ethyl)tetradecanamide (A-3-a)

TFA (2 ml, 26 mmol) was added to a solution of A-2-a (0.46 g, 1 mmol) in10 ml of DCM, and the mixture was stirred at RT for 3 h. The reactionmixture was concentrated, and the crude material was lyophilized toobtain a colorless oil that was dissolved in 10 ml of DCM. Bromoaceticanhydride (0.31 g, 1.2 mmol) was added, followed by DIEA (0.52 ml, 2.5mmol), and the mixture was stirred at RT for 3 h. The reaction mixturewas extracted with DCM and EtOAc, washed with 1% HCl, saturated NaHCO₃and brine, dried over Na₂SO₄, filtered and concentrated. The crudematerial was purified by flash column chromatography on silica gel witha gradient 20-50% EtOAc in petroleum ether with 5% methanol to obtain0.37 g of desired compound as a white solid (combined yield over twosteps, 78%). ¹H NMR (500 MHz; CDCl₃): δ 0.88 (t, J=6.5 Hz, 3H),1.25-1.32 (m, 20H), 1.62 (t, J=7.5 Hz, 2H), 2.18 (t, J=8.0 Hz, 2H), 3.47(dd, J=5.0, 10.0 Hz, 2H), 3.50 (dd, J=5.0, 10.0 Hz, 2H), 3.56-3.58 (m,2H), 3.59-3.62 (m, 2H), 3.63 (d, J=5.5 Hz, 5H), 3.88 (s, 2H), 5.92-5.93(m, 1H), 6.94 (s, 1H); 7.04-7.17 (m, 1H); 13C NMR (100 MHz; CDCl3): δ14.55, 23.12, 26.20, 29.76, 29.79, 29.95, 30.06, 30.08, 30.09, 30.11,32.35, 37.23, 69.84, 70.46, 70.83 (2), 166.06, 173.84; m/z (ESI+) 480.6(M+H).

(4Z,7Z,10Z,13Z,16Z,19Z)-N-(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)docosa-4,7,10,13,16,19-hexaenamide (A-3-b)

The title compound was prepared using analogous conditions as theprocedure for A-3-a. Yield 67%, brown oil. ¹H NMR (500 MHz; CDCl₃): δ0.96 (t, J=5.0 Hz, 3H), 2.07 (q, J=7.6 Hz, 2H), 2.23 (J=7.2 Hz, 2H),2.40 (dd, J=7.2, 13.8 Hz, 2H), 2.79-2.84 (m, 10H), 3.44-3.51 (m, 4H),3.55-3.62 (m, 4H), 3.63 (s, 4H), 3.88 (s, 2H), 5.78-5.42 (m, 12H), 5.94(s, 1H), 6.92 (s, 1H); m/z (ESI+) 559.8 (M+H).

(R)-N-(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide(A-3-c)

The title compound was prepared using analogous conditions as theprocedure for A-3-a, replacing A-2-a with A-2-c. Yield 87%, white solid.¹H NMR (500 MHz; CDCl₃): δ 0.83 (t, J=4.0 Hz, 3H), 0.85-0.94 (m, 6H),1.00-1.91 (m, 28H), 3.41-3.64 (m, 12H), 3.85-3.88 (m, 2H), 4.86-4.94 (m,1H), 6.05 (t, J=7.2 Hz, 1H), 7.04 (s, 1H); m/z (ESI+) 628.6 (M+H).

Synthesis of Peptides Derivatized with Fatty Acid or Bile Acid (A-4-a,A-4-b, or A-4-c)

The Cys peptide intermediate (1 equiv) is dissolved in DMSO and isreacted with any one of A-3-a, A-3-b, or A-3-c (2 equiv) dissolved inTHF, followed by addition of DIEA (3% by volume). Reaction completion isassessed by HPLC-MS. The reaction is quenched by addition of TFA to afinal pH of 4, and directly purified by preparative reverse-phase HPLCusing eluents (A) 0.05% TFA in water and (B) 0.05% TFA in acetonitrile,and the following gradient for eluent (B): 10% (1 min) 40-55% (10 min),flow rate 80 ml/min. The purified peptides are lyophilized, andstructure and purity are confirmed by analytical HPLC and electrospraymass spectrometry.

Example 4. Synthesis of Pegylated Lipid (Ether Linkage) ConjugatedRelaxin (Schemes B-1 and B-2)

A solution of 2-(2-(2-aminoethoxy)ethoxy)ethanol (100 mg, 0.671 mmol, 89μL) in 4.8 mL of acetonitrile/water (6:1) was treated with di-tert-butyldicarbonate (151 mg, 0.691 mmol), followed by 0.5 mL of 1 N NaOH (aq).After stirring at RT for 45 min, the organic solvent was removed invacuo, the residue was dissolved in saturated NH₄Cl (aq), and thedesired carbamate was extracted with EtOAc. Removal of EtOAc provided 57mg (34% yield) of tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy)ethylcarbamateas a colorless oil.

A solution of tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy)ethylcarbamate(68.5 mg, 0.275 mmol) and myristyl tosylate (101 mg, 0.275 mmol) in 1.4mL of t-amyl alcohol was treated with potassium tert-butoxide (61.6 mg,0.550 mmol) and potassium iodide (4.6 mg, 0.028 mmol). After heating to90° C. for 2 h, the reaction was allowed to cool to rt, quenched withsaturated NH₄Cl (aq), then extracted with EtOAc and dried over Na₂SO₄.Concentration and subsequent purification via flash columnchromatography on silica gel afforded 44 mg (36% yield) of tert-butyl2-(2-(2-(tetradecyloxy)ethoxy)ethoxy)ethylcarbamate as a colorless oil.

A solution of tert-butyl2-(2-(2-(tetradecyloxy)ethoxy)ethoxy)ethylcarbamate (26 mg, 0.058 mmol)in 1.2 mL of DCM was treated with 0.29 mL of trifluoroacetic acid. Afterstirring at rt for 50 min, the mixture was concentrated and re-dissolvedin 2 mL DCM. A 1-mL aliquot of this solution was taken into a separatevial, which was cooled to 0° C. and subsequently charged withbromoacetic anhydride (10.3 mg, 0.040 mmol) and DIEA (11 μL, 0.063mmol). After stirring at rt for 12 h, the reaction mixture was purifiedvia flash column chromatography to provide 7.5 mg (55% yield over 2steps) of2-bromo-N-(2-(2-(2-(tetradecyloxy)ethoxy)ethoxy)-ethyl)acetamide as anoff-white solid.

A solution of the Cys-peptide intermediate (1 equiv) in DMSO (4 mM) istreated with2-bromo-N-(2-(2-(2-(tetradecyloxy)ethoxy)ethoxy)-ethyl)acetamide (2.1equiv) in THF (5.4 mM) and DIEA (114 equiv). Upon reaction completion,the reaction mixture is purified by preparative reverse-phase HPLC toprovide the lipid conjugate (B-1-a).

tert-Butyl 2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethylcarbamate (B-2-a)

A solution of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol (400 mg, 2.07mmol, 0.33 mL) in 15 mL of acetonitrile/water (6:1) was treated withdi-tert-butyl dicarbonate (603 mg, 2.77 mmol), followed by 2.8 mL of 1 NNaOH (aq). After stirring at RT for 45 min, the organic solvent wasremoved in vacuo, the residue was dissolved in saturated NH₄Cl (aq), andthe desired carbamate was extracted with EtOAc. Removal of EtOAcprovided the crude carbamate as a colorless oil.

This oil was dissolved in DCM and treated with p-toluenesulfonylchloride (1.18 g, 6.21 mmol) and pyridine (0.84 mL, 10.4 mmol). Afterstirring at 40° C. for 12 h, the mixture was diluted with DCM and washedwith 1N HCl (2×10 mL), H₂O (10 mL), and brine (10 mL), then dried overNa₂SO₄ and concentrated. Purification via flash column chromatography onsilica gel gave 330 mg (36% yield over 2 steps) of the tosylate as acolorless oil.

A solution of the tosylate (203 mg, 0.453 mmol) in 3.1 mL of anhydrousacetone was treated with LiBr (385 mg, 4.53 mmol). After stirring at 60°C. for 8 h, the solvent was removed and the resulting residue wasdissolved in EtOAc. The organic mixture was washed with water, driedover MgSO₄, filtered, and concentrated. Purification via flash columnchromatography on silica gel gave 126 mg (78% yield) of the titlecompound as a colorless oil.

2-((3S,8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)ethanol(B-2-b)

A solution of cholesterol (1.50 g, 3.89 mmol) in 4 mL of DCM was treatedwith p-toluenesulfonyl chloride (1.49 g, 7.78 mmol), pyridine (4 mL),and DMAP (94.9 mg, 0.780 mmol). After stirring at RT for 12 h, themixture was diluted with DCM and washed with 1N HCl (2×5 mL), H₂O (5mL), and brine (5 mL), then dried over Na₂SO₄ and concentrated in vacuo.Recrystallization from chloroform and methanol gave 1.66 g (79% yield)of the tosylate intermediate as a white solid.

A microwave vial charged with the tosylate intermediate (500 mg, 0.926mmol) in 7.7 mL of 1,4-dioxane was treated with 2.6 mL of ethyleneglycol. After heating to 160° C. by microwave irradiation for 10 min,the solvent was removed, and the residue was dissolved in chloroform andwashed with saturated NaHCO₃ (5 mL), H₂O (5 mL), and brine (5 mL), thendried over Na₂SO₄ and concentrated in vacuo. Purification of the crudematerial by flash column chromatography using silica gel provided 270 mg(68% yield) of the title compound as a white solid.

2-Bromo-N-(14-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6,9,12-tetraoxatetradecyl)acetamide(B-2-c)

A solution of B-2-b (219 mg, 0.509 mmol) in 2.5 mL of THF was treatedwith sodium hydride (60% dispersion in mineral oil, 20.4 mg, 0.509 mmol)and stirred for 30 min. The mixture was cooled to 0° C. and treated withB-2-a (139 mg, 0.392 mmol) in 2.5 mL of THF. After heating to 40° C. for6 h, the reaction was quenched with saturated NH₄Cl (aq) and extractedwith EtOAc. Purification of the crude material by flash columnchromatography using silica gel provided 101 mg (36% yield) of the etherintermediate as a colorless oil.

A solution of the ether intermediate (74 mg, 0.105 mmol) in 2.1 mL ofDCM was treated with 0.53 mL of TFA. After stirring at RT for 40 min,the mixture was concentrated in vacuo and dissolved in 10 mL DCM. A 4-mLaliquot of this solution was taken into a separate vial, concentrated toa 1-mL volume, cooled to 0° C., and charged with bromoacetic anhydride(13.6 mg, 0.053 mmol) and DIEA (15 μL, 0.088 mmol). After stirring at RTfor 12 h, the reaction mixture was directly purified by flash columnchromatography using silica gel to give 12.6 mg (41% over 2 steps) ofthe title compound as an colorless oil.

Lipid conjugate B-2-d is prepared in an analogous manner as lipidconjugate B-1-a of Example 4, Scheme B-1.

Example 5. Preparation of Br-FA₁

Step A. Tert-butyl (2-(2-(2-tetradecanamidoethoxy)ethoxy)ethyl)carbamate(1-1)

Myristic acid (0.46 g, 2 mmol) was dissolved in 5 mL of DMF. HATU (0.8g, 2.1 mmol) and DIPEA (0.4 mL, 2.2 mmol) were added followed by theaddition of Boc-NH-PEG2-COOH (0.5 g, 2 mmol). The reaction mixture wasthen stirred for 6 h, and the solvent was removed. The product wasextracted with EtOAc (3×15 mL). The organic layer was successivelywashed with sat. NaHCO₃, cooled HCl (1 M) and brine, dried over Na₂SO₄,filtered, and concentrated. Purification by flash column chromatographyon silica gel provided 0.81 g of desired compound as white solids in 90%product yield. ESI-MS: calcd MW 458.4. found 459.6 [M+1]⁺.

Step B. N-(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)tetradecanamide(Br-FA₁)

A solution of 1-1 (0.23 g, 0.5 mmol) in DCM (10 mL) was treated with TFA(2 mL) for 2 h. The mixture was concentrated and followed by theaddition of bromoacetic anhydride (0.14 g, 0.55 mmol), DIPEA (0.17 mL, 1mmol) in 10 mL of DCM at 0° C. The reaction mixture was then stirred for2 h, and the solvent was removed. The product was extracted with EtOAc(3×15 mL). The organic layer was successively washed with sat. NaHCO₃,cooled HCl (1 M) and brine, dried over Na₂SO₄, filtered, andconcentrated. Purification by flash column chromatography on silica gelprovided 0.2 g of the title compound as white solids in 83% productyield. ESI-MS: calcd MW 479.5. found 480.4 [M+1]⁺.

Example 6. Solid-Phase Synthesis of Br-FA₂

Fmoc-Lys(ivDde)-OH (60 mg, 100 μmol) was attached to 2-chlorotritylchloride resin (Novabiochem) (100 mg, 80 μmol) by mixing the amino acid,the resin, and DIPEA (70 μL, 400 μmol) in 5 mL of DMF and stirring for30 min. The resin was then washed with DMF (3×), DCM (3×) and treatedwith CH₃OH/DCM/DIPEA (8:1:1) for 10 min to cap the unreacted tritylchloride sites, dried under vacuum, and stored in a desiccator.

To this resin (50 mg, 40 μmol) was added piperidine in DMF (20%, 5 mL).The mixture was shaken for 1 min and drained. Another 5 mL of 20%piperidine was added and this time the mixture was shaken for 15 min.Positive ninhydrin test was observed. The resin was then washed asdescribed above.

The resin was then treated with octadecanedioic acid mono-tert-butylester (AstaTech) (74 mg, 200 μmol) using coupling reagent HATU (76 mg,200 μmol), and DIPEA (35 μL, 200 μmol) in DMF (5 mL) for 2 h or repeateduntil a negative ninhydrin test was observed. After washing with DMF andDCM, the resin was treated with 2% hydrazine in DMF (5 mL, 2×5 min).Positive ninhydrin test was observed. The resin was then washed asdescribed above.

The resin was then treated with Fmoc-PEG2-propionic acid (QuantaBioDesign) (80 mg, 200 μmol) using HATU (76 mg, 200 μmol), and DIPEA (35μL, 200 μmol) in DMF (5 mL) for 2 h or repeated until a negativeninhydrin test was observed. The resin was then washed as describedabove. Then the protecting group is removed and the above steps arerepeated.

The resin was then treated with bromoacetic anhydride (55 mg, 200 μmol),and DIPEA (35 μL, 200 μmol) in 2 mL of DCM and stirring for 30 min.After washing with DCM (3×), the product was cleaved from the resinusing 5 mL of 10% TFA in DCM containing 10% H₂O and 10%triisopropylsilane for 1 h. After cleavage, TFA was removed underreduced pressure. The resulting yellow residue was washed several timeswith cold diethyl ether and was finally dried to a crude product asyellow powder under nitrogen flow.

Attempts to dissolve 50 mg of the crude product in up to 0.5 mL of 50%CH₃CN in water (0.1% TFA) have been unsuccessful. Therefore, as analternative, the crude peptide, (50 mg) was dissolved in DMSO (0.1 mL)and this solution was diluted to a final volume of 0.5 mL with 50%CH₃CN-water. The solution was filtered. The filtered solution was loadedonto the preparative HPLC column (Phenomenex, Prep C18, 300A, 50×250 mm)equilibrated with 10% CH₃CN (0.1% TFA) in water (0.1% TFA), and thecolumn was eluted with 10% CH₃CN (0.1% TFA) in water (0.1% TFA) to washDMSO from the column. The composition of the eluent then was ramped to35% CH₃CN-water (0.1% TFA) over 1 min, and a linear gradient wasinitiated at a rate of 0.5%/min of CH₃CN (0.1% TFA) into water (0.1%TFA) and run for 50 min. Eluted fractions were checked for purity on ananalytical reversed phase C18 column (Phenomenex, C18, 120A, 4.6×50 mm)and fractions containing the product in >95% purity were combined andfreeze-dried to afford the title compound (15 mg, 41% yield). Themolecular weight of product was analyzed by ESI-MS: calcd MW 880.9.found 881.3 [M+1]⁺, 882.6 [M+2]⁺.

Example 7. Solid-Phase Synthesis of Br-FA₃

The title compound was prepared in an analogous manner as Br-FA₂ bysubstituting 2-chlorotrityl chloride resin with rink amide resin.Purification by preparative HPLC and lyophilization gave the titlecompound as a white powder with greater than 95% purity in about 45%product yield. The molecular weight of peptide was analyzed by ESI-MS:calcd MW 881.0. found 882.2 [M+1]⁺, 883.7 [M+2]⁺.

Example 8. Preparation of Br-FA₄

A solution of tetradecylamine (107 mg, 0.5 mmol) in DCM (10 mL) wastreated with bromoacetic anhydride (0.14 g, 0.55 mmol), DIPEA (100 μL,0.55 mmol) at 0° C. The reaction mixture was then stirred for 2 h, andthe solvent was removed. The product was extracted with EtOAc (3×15 mL).The organic layer was successively washed with sat. NaHCO₃, cooled HCl(1 M) and brine, dried over Na₂SO₄, filtered, and concentrated.Purification by flash column chromatography on silica gel provided 145mg of the title compound as white solids in 87% product yield. ESI-MS:calcd MW 334.3. found 335.6 [M+1]⁺.

Example 9. Preparation of Br-FA₅

Step A. Tert-butyl(9,20-dioxo-3,6,13,16-tetraoxa-10,19-diazatritriacontyl)carbamate (1-2)

A solution of 1-1 (115 mg, 0.25 mmol) in DCM (10 mL) was treated withTFA (2 mL) for 2 h. The mixture was concentrated and followed by theaddition of Boc-NH-PEG2-acid (70 mg, 0.25 mmol), HATU (95 mg, 0.25 mmol)and DIPEA (90 μL, 0.5 mmol) in 5 mL of DMF. The reaction mixture wasthen stirred for 2 h, and the solvent was removed. The product wasextracted with EtOAc (3×15 mL). The organic layer was successivelywashed with sat. NaHCO₃, cooled HCl (1 M) and brine, dried over Na₂SO₄,filtered, and concentrated. Purification by flash column chromatographyon silica gel provided 122 mg of desired compound as white solids in 77%product yield. ESI-MS: calcd MW 617.9. found 618.4 [M+1]⁺.

Step B.N-(1-bromo-2,12-dioxo-6,9,16,19-tetraoxa-3,13-diazahenicosan-21-yl)tetradecanamide(Br-FA₅)

A solution of 1-2 (122 mg, 0.2 mmol) in DCM (10 mL) was treated with TFA(2 mL) for 2 h. The mixture was concentrated and followed by theaddition of bromoacetic anhydride (52 mg, 0.2 mmol), DIPEA (70 μL, 0.4mmol) in 5 mL of DCM at 0° C. The reaction mixture was then stirred for2 h, and the solvent was removed. The product was extracted with EtOAc(3×15 mL). The organic layer was successively washed with sat. NaHCO₃,cooled HCl (1 M) and brine, dried over Na₂SO₄, filtered, andconcentrated. Purification by HPLC provided 88 mg of the title compoundas white solids in 69% product yield. ESI-MS: calcd MW 638.7. found639.4 [M+1]⁺.

Example 10. Preparation of Br-FA₆

Step A. tert-Butyl (2-(2-(2-tetradecanamidoethoxy)ethoxy)ethyl)carbamate(1-3)

Octadecanedioic acid mono-tert-butyl ester acid (0.18 g, 0.5 mmol) wasdissolved in 5 mL of DMF. HATU (0.2 g, 0.55 mmol) and DIPEA (0.1 mL,0.55 mmol) were added followed by the addition of Boc-NH-PEG3-NH₂ (0.12g, 0.5 mmol). The reaction mixture was then stirred for 6 h, and thesolvent was removed. The product was extracted with EtOAc (3×10 mL). Theorganic layer was successively washed with sat. NaHCO₃, cooled HCl (1 M)and brine, dried over Na₂SO₄, filtered, and concentrated. Purificationby flash column chromatography on silica gel provided 0.28 g oftert-butyl (2-(2-(2-tetradecanamidoethoxy)ethoxy)ethyl)carbamate aswhite solids in 87% product yield. ESI-MS: calcd MW 644.5. found 645.5[M+1]⁺.

Step B. 1-Bromo-2,16-dioxo-6,9,12-trioxa-3,15-diazatritriacontan-33-oicacid (Br-FA₆)

A solution of 1-3 (65 mg, 0.1 mmol) in DCM (2 mL) was treated with TFA(4 mL) for 4 h. The mixture was concentrated and followed by theaddition of bromoacetic anhydride (26 mg, 0.1 mmol), DIPEA (35 μL, 0.2mmol) in 5 mL of DCM at 0° C. The reaction mixture was then stirred for2 h, and the solvent was removed. The product was purified by HPLCprovided 35 mg of the title compound as white solids in 70% productyield. ESI-MS: calcd MW 608.3. found 609.4 [M+1]⁺.

Example 11. Solid Phase Synthesis of Br-FA₇

The title compound was prepared in an analogous manner as Br-FA₂ bysubstituting Fmoc-Lys(ivDde)-OH and Fmoc-PEG2-propionic acid withmono-Fmoc ethylene diamine hydrochloride and[2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid, respectively. Purificationby preparative HPLC and lyophilization afforded the title compound as awhite powder with greater than 95% purity in about 25% product yield.The molecular weight of peptide was analyzed by ESI-MS: calcd MW 896.9.found 898.1 [M+1]+.

Example 12. Preparation of Br-FA8

The title compound was prepared in an analogous manner as Br-FA₂ bysubstituting octadecanedioic acid mono-tert-butyl ester with palmiticacid. Purification by preparative HPLC and lyophilization afforded thetitle compound as a white powder with greater than 95% purity in about29% product yield. The molecular weight of peptide was analyzed byESI-MS: calcd MW 822.4. found 823.6 [M+1]⁺.

Example 13. Preparation of Inclusion Bodies

Cells expressing relaxin analogs were spun down to produce a cellpellet. The cell pellet was re-suspended in lysis buffer (100 mM Tris,pH 8, 100 mM NaCl, 1% Triton-X100, 2 mM EDTA) and lysed by passingthrough French pressure cell operated at 18,000 psi for at least 3 timeswhile chilling the cell suspension to 4° C. after each pass. Lysed cellsuspension was clarified by centrifuging for 45 min at 15,000 rpm, 4° C.The pellet contains intact cells, cellular debris along with inclusionbody protein. So the pellet was washed multiple times to isolate theinclusion body protein from the rest, briefly, after decanting thesupernatant, the pellet was re-suspended in lysis buffer using a tissuehomogenizer and subjected to centrifugation, this procedure was repeatedat least thrice. To remove the Triton-X 100 from the pellet, the pelletwas washed at least twice with wash buffer (100 mM Tris, pH 8, 100 mMNaCl, 1% Triton-X100, 2 mM EDTA); washing of pellet was performed byresuspension through homogenization followed by centrifugation. Thepellet was then directly used for in vitro refolding of the relaxinanalog without any further purification.

Example 14. Refolding of Relaxin Analogs

The cell pellet from Example 13 was used for in vitro refolding ofrelaxin analogs. Refolding of the relaxin analogs was carried usingoxidized/reduced glutathione. Inclusion bodies were dissolved in 8Mguanidine hydrochloride (GdnHCl). 40 mL of GdnHCl was used to dissolveinclusion body pellet isolated from 1 L E. coli culture. Aftersolubilizing the protein, the insoluble portion was pelleted bycentrifuging at 15000 rpm for 30 min. The solubilized protein was thenconcentrated using 3,000 MW cutoff Amicon Ultra (Fisher Scientific)centrifugal filter unit to 10-15 mL. The denatured protein was thendiluted in to 300 mL of refolding buffer (440 mM Arginine, 55 mM Tris,pH 8.2 containing 2 mM GSH, 10 mM GSSG). The refolding was continued for1-2 hours and quenched by adding TFA to pH 2. The precipitate was thencentrifuged to a pellet and the supernatant was transferred in tocentrifugal unit and concentrated to 2-3 mg/mL. The concentrated proteinmixture was then purified by semi-preparative HPLC using PhenomenexJupiter C5 column (250×4.6 mm) with the flow rate set at 4.0 mL/min. Thefractions were analyzed by LC-MS for the desired protein and then pooledand lyophilized.

Example 15. Conjugation of Lipid to Relaxin

All the enzymatic processing (Trypsin/Cp-B or TEV protease) wereperformed after capping cysteine, as the free thiol in relaxin wasobserved to cause scrambling at basic pH where enzymatic digestion aregenerally performed. The lyophilized protein from Example 14 wasdissolved in 0.25% TFA. Pegylated lipid was dissolved in ACNacetonitrile/100 mM NH₄HCO₃ buffer (1:1), pH 8.5. Relaxin was then addedto the mixture and the reaction proceeded at room temperature for 2hours; the progress of the reaction was checked by LC-MS. Aftercompletion of reaction, the mixture was then lyophilized.

Example 16. Enzymatic Conversion of Relaxin Precursor into Two-ChainPrecursor

The lyophilized powder from Example 15 was resuspended in 50 mM Trisbuffer containing 1 mM CaCl₂, pH8. Trypsin (1:100) was added to thismixture, and the mixture was incubated for 1 hour at room temperature.After 1 hour, carboxy peptidase-B (10 units for 1 mg of protein) wasadded in to the mixture and the progress of the cleavage was monitoredby LC-MS analysis. After the complete cleavage, the pH of the mixturewas brought to 4 by adding acetic acid. The mixture was lyophilized andpurified semi-preparative HPLC.

Example 17. Cleavage of His-Tag Through TEV Protease

The lyophilized protein of Example 16 was re-dissolved in PBS buffercontaining 3 mM GSH: 0.5 mM GSSG. Tev protease was added in to themixture and incubated overnight at room temperature. The cleavage of tagwas confirmed by LC-MS analysis.

Example 18. Gene Construction and Expression of Toxin-550 Analogs

The inserts were prepared through polymerase cycling assembly, whereforward and reverse primers were annealed to generate the DNA fragmentwhich was further enriched by forward and reverse primers. The amplifiedfragment was then digested with restriction digestion enzymes (BamI/Hind-III). The prepared insert was then ligated into a vector with aPET backbone, the encoded DNA fragment was then confirmed by DNAsequencing. Toxin-550 constructs (pET/Txin-550) were transformed into E.coli strain BL21 (DE3). The transformed E. coli cells were cultured interrific broth (with 50 μg/ml kanamycin) to OD600 nm=1.0 at 37° C. withvigorous shaking (250 rpm). To induce expression of the proteins, IPTGstock solution (1M) was added to the final concentration of 0.5 mM, andthe cells were continuously incubated with shaking at 250 rpm at 26° C.for 24 h. On the following day, cells were harvested by centrifugation(6000 rpm, 15 min).

Preparation of Inclusion Bodies

A cell pellet was re-suspended in lysis buffer (100 mM Tris, pH 8, 100mM NaCl, 1% Triton-X100, 2 mM EDTA) and lysed by passing through Frenchpressure cell operated at 18,000 psi for at least 3 times while chillingthe cell suspension to 4° C. after each pass. Lysed cell suspension wasclarified by centrifuging for 45 min at 15,000 rpm, 4° C. The pelletcontained intact cells, cellular debris along with inclusion bodyprotein. The pellet was washed multiple times to isolate the inclusionbody protein from the rest of the cells and other cellular debris.Briefly, after decanting the supernatant, the pellet was re-suspended inlysis buffer using a tissue homogenizer and subjected to centrifugation,this procedure was repeated at least thrice. To remove the Triton-X 100from the pellet, the pellet was washed at least twice with wash buffer(100 mM Tris, pH 8, 100 mM NaCl, 1% Triton-X100, 2 mM EDTA), washing ofpellet was performed by suspension through homogenization followed bycentrifugation. The pellet was then directly used for in vitro refoldingwithout any further purifications.

Example 19. Refolding of Toxin-550 Analogs

Refolding was carried using oxidized/reduced glutathione. Inclusionbodies (as in Example 18) were dissolved in 8M guanidine hydrochloride(GdnHCl). 10 mL of GdnHCl was used to dissolve the inclusion body pelletisolated from 1 L E. coli culture. After solubilizing the protein,another 10 mL of Tris-HCL buffer (50 mM) was added and the insolubleportion was pelleted by centrifuging at 15000 rpm for 30 min. Thesolubilized supernatant denatured protein was then diluted in to 200 mLof refolding buffer. Various refolding buffers that were used to dilutethe solubilized supernatant denatured protein are shown in the tablebelow:

Refolding Buffer GSH:GSSG 0.1M Tris, pH 8.2 1:10 0.1M Tris, pH 11 1:100.1M NH₄HCO₃, pH 8 1:10 0.1M NH₄OAC 1:10 2M NH₄OAC 1:10 2M (NH₄)₂SO₄1:10

FIG. 10 shows the disulfide bond pattern for the Toxin-550 peptide(wild-type) and an exemplary in vitro folding pathway of cysteine-knotToxin-550 peptide, the middle structure depicting a two-sulfide bondintermediate, in which a disulfide bond is formed between cysteine I andcysteine IV and between cysteine II and cysteine V.

Various refolding trails were employed, with most of the refoldingbuffer conditions leading to a two-disulfide bond intermediate as majorproduct, with the exception of 2 M (NH₄)₂SO₄ refolding buffer, where thethree disulfide bond product is observed in 4 hours. The three disulfidebond product contains a Toxin-550 peptide with a disulfide bond formedbetween cysteine I and cysteine IV, between cysteine II and cysteine V,and between cysteine III and cysteine VI.

Example 20. Conjugation of Lipid to Toxin-550

The refolded toxin-550 was then buffer exchanged with PBS, 2% TFA toremove the excess glutathione before conjugation with lipids. Pegylatedlipid was dissolved in ACN acetonitrile/100 mM NH₄HCO₃ buffer (1:1), pH8.5. Toxin was then added to the mixture and the reaction proceeded atroom temperature for 2 hours; the progress of the reaction was checkedby LC-MS. After completion of reaction, the mixture was then lyophilizedand purified by RP-HPLC using C18-phenomenex column. The lipidconjugates comprising a lipid attached to toxin-550 are shown in FIG.16B as entries 11-13. The brackets depict the disulfide bonds formedbetween cysteine residues in the lipid conjugates

Example 21. Conjugation of Lipid to Oxyntomodulin and ExenatideDerivatives

The oxyntomodulin Cys38 (e.g., Oxm-Cys38, SEQ ID NO: 24) and exenatideCys40 (e.g., Ex4-Cys40, SEQ ID NO: 25) were chemically synthesized usingsolid-phase peptide synthesis. Pegylated lipid was dissolved in ACNacetonitrile/100 mM NH₄HCO₃ buffer (1:1), pH 8.5. The cysteine mutantwas dissolved in ACN acetonitrile/100 mM NH₄HCO₃ buffer and then addedto the pegylated lipid mixture and the reaction proceeded at roomtemperature for 2 hours. Progress of the reaction was checked by LC-MS.After completion of reaction the mixture was then lyophilized andpurified by RP-HPLC using C18-phenomenex column.

Example 22. Conjugation of XTEN to Relaxin Derivative

The lyophilized protein, refolded relaxin with free cysteine wasre-suspended in 0.25% TFA in water and acetonitrile mixture.Iodoacetylated XTEN 288 or 864 (1.2 equivalents; iodoacetylated atN-terminus) was added and then the pH was adjusted to 8.5 using 1Mammonium bicarbonate. The reaction proceeded at room temperature for 2hours; the progress of the reaction was checked by LC-MS. Aftercompletion of reaction, the mixture was then buffer exchanged prior topurification via anion-exchange chromatography (Mono-Q). The desiredfractions were then collected and underwent cleavage of mini-C-chainfollowing similar procedures as exemplified in Example 16. The cleavagereaction was quenched and subjected to RP-HPLC to purify theXTEN-relaxin conjugates. Select data shown in Table 3.

The following compounds in Table 1 were or are prepared using analogousprocedures. Additional examples are shown in FIGS. 16A-B and 17.

TABLE 1 en- try Structure*  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43XCH₂C(O)-SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR (core sequence disclosedas SEQ ID NO: 66) 44XCH₂C(O)-SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTESASR (core sequence disclosed as SEQ ID NO:67) *X can be Cl, I, Br, maleimide, or an amino acid which is part ofthe peptide (TA); Y can be OH or an amino acid which is part of thepeptide (TA); Z can be S which is part of an amino acid which is part ofthe peptide (TA) or S which is part of a lipid derivative (to form adisulfide).

Example 23. Measuring Bioactivity of Ex4 and Oxm Peptides

Ex4 is a GLP1R agonist, and Oxm is a GLP1R and GCGR dual agonist. Invitro activity was determined based on the stimulation of adenylatecyclase activity in Hek293 cells stably expressing the GLP1R and GCGRreceptor. Stable GCGR and GLP1R receptor expressing cells weremaintained in Dulbecco's modified Eagle's medium/F-12 media supplementedwith 200 ng/ml Zeocin (Invitrogen). A cyclic AMP responsive elementdriven luciferase (CRE-Luc) reporter line was generated by lentiviraltransduction of these receptor overexpressing HEK293T cells with CRE-Luclentivirus (Qiagen) and selected with puromycine (2 μg/mL) for twoweeks. A reporter gene assay was used to detect cAMP levels and GLP1Rand GCGR activation by Ex-4 or Oxm.

Assay detail: HEK293T cells expressing GLP1R and CRE-Luc were seeded ata density of 5×10³ cells per well in 504 of Dulbecco's modified Eagle'smedium/F-12 medium containing 10% FBS in 384-well solid bottom whiteplates. Cells were pre-incubated at 37° C. for overnight. Differentconcentrations of peptides (from 0.001 nM to 100 nM) were added intriplicate, incubated for 18 hours and luciferase activity was detectedby adding 104 of Bright Glo (Promega). Luminescence was recorded onEnvision (Perkin Elmer). EC₅₀ was calculated after non-linear curvefitting. For GCGR activity, a reporter cell the with GCGR and CRE-Lucwas employed. Select data are shown in Table 3.

Example 24. Measuring Bioactivity of Toxin Peptides

Toxin-550 peptides were assayed on the IonFlux HT using standard runningconditions for measurement of NaV 1.7 current. Briefly, HEK293 cellsoverexpressing the alpha and beta-1 subunits of human Nav1.7 (PrecisION,Millipore) were trypsinized, brought up in serum-free media and allowedto shake at room temperature for 30 minutes. Compounds were diluted intostandard extracellular Ringer's solution (ECS) at desired concentration.Cells were washed once with ECS and then brought up in ECS at aconcentration of 4 million cells/mL. IonFlux HT plate was set upaccording to standard protocols.

NaV 1.7 current was measured on the IonFlux according to the followingvoltage step protocol:

1. The cells were held at −90 mV with a period of 10 seconds.

2. The cells were stepped from −90 mV to −120 mV for 100 ms.

-   -   a. then to −10 mV for 30 ms and    -   b. then back to −90 mV for 30 ms.

Baseline current was allowed to stabilize for 5 minutes, then a controlbaseline (ECS only) was established. Compound was applied for 20minutes, and then a positive control (peptide NaV1.7 blocker) wasapplied.

Leak subtraction was then performed on the data and the resultingcurrents are analyzed as follows:

-   -   1. Cursor subtraction (baseline-peak) was performed for all        current sweeps to determine the amplitude of current (I).    -   2. ECS baseline current for each patch was taken as maximum        current (I_(max)).    -   3. For a given time (e.g. 10 minutes post-compound addition),        percent block was calculated as I/I_(max).

Select data are shown in Table 3.

Example 25. In Vivo Pharmacokinetic (PK) Study for Half-Life Extension

Lipid conjugated relaxin or other therapeutic agents are dosed in miceusing sc, po, iv dosing. Plasma levels of TA are determined by ms, lgr7,glp1r and/or gcgr activation at different time points (e.g., i.v. PK 5min, 0.5 h, 1 h, 2 h 4 h, 8 h, 24 h, 48 h, 96 h & 144 h and s.c. PK. 15min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h and 144 h). Select PKdata are shown in FIGS. 11, 12, and 15, and Table 2.

TABLE 2 Activity, Half- fold drop life Mass Mass compared (hours) EntryName expected found to relaxin i.v., s.c. 1 Pro-Relaxin-B-D1A 8183.78184.12 10 2 Relaxin-B-D1A 5936.15 5936.92 0 0.5, 0.8 3Relaxin-B-D1AS29C-FA₁ ^(a) 6349.98 6350.52 0 0.5, 1.2 4Relaxin-B-D1AS29C-FA₂ 6752.87 6753.27 70 5 Relaxin-B-D1AS29C-FA₃ 6751.906752.49 30 6 Relaxin-B-D1AS29C-FA₄ 6205.14 6205.62 170 7Relaxin-B-D1AS29C-FA₅ 8 Relaxin-B-D1AS29C-PEG20K ND 30 9 Relaxin-B-D1A,A-Q1C-FA₁ ^(b) 6309.10 6309.87 10 10 Relaxin-B-D1A, A-Q1C-FA₂ 6711.726712.45 15 3.5, 7.8 11 Relaxin-B-D1A, A-Q1C-FA₃ 6710.75 6711.33 25 12Relaxin-B-D1A, A-Q1C-PEG20K 3.0, 7.1 13 Relaxin-B-D1A, A-A5C-FA₁ ND 14Relaxin-B-D1A, A-A5C-FA₂ 6768.95 6769.36 25 15 Relaxin-B-D1A, A-A5C-FA₃6767.98 6768.42 15 16 Relaxin-B-D1A, A-A5C-PEG20K 17 Relaxin-B-D1C-FA₁6366.20 6366.82 3 18 Relaxin-B-D1C-FA₂ 6768.82 6769.34 18 19Relaxin-B-D1C-FA₃ 20 Relaxin-B-D1C-PEG-20K 21 Relaxin-B-D1A, A-R18C-FA₁6281.05 6281.79 15 22 Relaxin-B-D1A, A-R18C-FA₂ 6683.67 6684.26 30 23Relaxin-B-D1A, A-R18C-FA₃ 6682.70 6683.27 30 24 Relaxin-B-D1A,A-R18C-PEG-20K ND 25 Relaxin-B-D1AM25KM4K, A- 6278.29 6279.01 25Q1AH12K, B-S29C-FA₁ 26 Relaxin-B-D1AM25KM4K, A- 6680.91 6681.42 150Q1AH12K, B-S29C-FA₂ 27 Relaxin-B-D1AM25KM4K, A- 6679.94 6680.63 150Q1AH12K, B-S29C-FA₃ 28 Relaxin-B-D1AM25KS29C-FA₂ 6749.84 6750.32 48 29Relaxin-B-D1AM4KS29C-FA₂ 6749.84 6750.58 40 30 Relaxin-A-H12K,B-D1AS29C-FA₂ 6743.91 6744.60 16 31 Relaxin-B-D1AM25KM4K, A- 6623.976624.48 8 Q1AH12A, B-S29C-FA₂ 32 Relaxin-B-D1AS29C-XTEN288 32236.132236.8 20 1.5, 4.5 ^(a)“B-” denotes B-chain. ^(b)“A-” denotes A-chain

TABLE 3 En- Mass Mass Activity try Name expected found (nM) 1Tev-Relaxin-single-chain 8841.1 8841.3 14 2 GSGG-Relaxin-single-chain6817.9 6818.0 1 (“GSGG” disclosed as SEQ ID NO: 71) 3Tev-Relaxin-B-S29C- 8857.2 8857.3 11 single-chain 4Tev-Relaxin-B-S29C-FA₁- 9255.8 9255.8 29 single-chain 5 Tev-Relaxin-Cchain 20411.4 20495.5 6 6 Tev-Relaxin 8130.4 8214.5 2 7Tev-Relaxin-B-S29C-FA₁- 20826.0 20925.9 21 C-Chain 8Tev-Relaxin-B-S29C-FA₁ 8544.9 8645.0 1 9 Ex4-Cys40-FA₁ 4688.6 1173.10.14 ([M + 4H]⁴⁺); (GLP-1R) 938.6 ([M + 5H]⁵⁺) 10 Oxm-Cys38-FA₁ 4950.81238.7 100 ([M + 4H]⁴⁺); (GLP-1R) 991.2 and ([M + 5H]⁵⁺) 50 (GCGR) 11550- 3947.1 3947.3 87% block at 100 nM 12 550-4-GSGG (“GSGG” 4310.64310.2 ND disclosed as SEQ ID NO: 71) 13 550-4-GSCGG-FA₁ 4709.4 4709.185% block (“GSCGG” disclosed as at 100 nM SEQ ID NO: 72) 14 550-GSGG(“GSGG” 4207.5 4206.9 ND disclosed as SEQ ID NO: 71) 15 550-3-GSGG(“GSGG” 4310.7 4310.2 ND disclosed as SEQ ID NO: 71) 16 550-3-GSGG-FA₁4709.3 4709.1 ND (“GSGG” disclosed as SEQ ID NO: 71) 17 550-3-GGS 4253.94253.1 ND 18 550-3-GGS-FA₁ 4652.7 4652.3 ND

Example 26. Pharmacodynamic (PD) Study and In Vivo Efficacy in AcuteHeart Failure Model

After determination of the PK, appropriate dose regimen are determinedand lipid conjugated relaxin are dosed using sc, po or iv to evaluatethe efficacy of lipid conjugated relaxin in efficacy models for bloodpressure, urine flow and/or ligament elongation. Also, lipid conjugatedrelaxin is evaluated in bleomycin induced fibrosis model in lung andliver and/or in diabetic wound healing models in old diabetic zucker fatrats.

Example 27. PD Study Using Mouse Interpubic Ligament Bioassay

The bioactivities of relaxin analogs were determined in CD1 mice byemploying the well-established and highly specific mouse interpubicligament bioassays for relaxin as described by Steinetz et al.[Endocrinology (1960) 67:102-115]. Female CD1 virgin mice at 3-4 weeksold were chosen from the study. Animals were group-housed undercontrolled temperature (25° C.) and photoperiod (12:12-hour light-darkcycle) conditions, and given unrestricted access to standard diet andtap water (or specified drinking solution). On day 1 of treatment, micewere s.c. injected with 10 μg estradiol cypionate in 0.1 ml sesame oil.On day 8, mice were divided into different groups: vehicle control, wtrelaxin (dissolved in a suspension of 1% benzopurpurine 4B in PBSbuffer) and relaxin analogs (dissolved in 20 mM NH₄Ac buffer, pH 6.5).Wt relaxin or relaxin analogs were administered at different dosethrough s.c. injection. At different time after relaxin injection, micewere euthanized and the length of interpubic ligaments was determinedusing caliper measurement. Select data are shown in FIGS. 13 and 14.

Example 28. Efficacy and Safety of Lipid Conjugated Relaxin for theTreatment of Acute Heart Failure

Purpose: Different doses of lipid conjugated relaxin are compared toplacebo to determine efficacy and safety for the treatment of patientshospitalized with acute heart failure.

Condition Intervention Phase Heart Failure, Congestive Drug: RelaxinPhase 1 Drug: Placebo Phase 1

Study Type: Interventional

Study Design: Allocation: Randomized

Endpoint Classification: Safety/Efficacy Study

Intervention Model: Parallel Assignment

Masking: Double Blind (Subject, Caregiver, Investigator, OutcomesAssessor)

Primary Purpose: Treatment

Primary Outcome Measures: Relief of dyspnea in acute heart failure [TimeFrame: Up to day 5] [Designated as safety issue: No]

Secondary Outcome Measures:

Days alive and out of hospital [Time Frame: Up to day 60] [Designated assafety issue: No]

CV death or rehospitalization due to heart failure or renal failure[Time Frame: Up to day 60] [Designated as safety issue: No]

Arms Assigned Intervention Placebo Comparator: Placebo Drug: Placebo 48hour iv infusion of placebo Intravenous infusion for 48 h Experimental:Lipid Conjugated Relaxin Drug: Lipid Conjugated Relaxin 48 hour ivinfusion of lipid conjugated Intravenous infusion for 48 h at relaxin at30 ug/kg/day 30 ug/kg/day

DETAILED DESCRIPTION

This is an international, randomized, double-blind, placebo-controlled,Phase II/III trial of intravenous recombinant relaxin for the treatmentof signs and symptoms in patients hospitalized for acute decompensatedheart failure. The Phase II pilot study has completed; the Phase IIImain portion of the trial is ongoing.

Eligibility

Ages Eligible for Study: 18 Years and older

Genders Eligible for Study: Both

Accepts Healthy Volunteers: No

Criteria

Inclusion Criteria:

Hospitalized for acute heart failure

Dyspnea at rest or with minimal exertion

Pulmonary congestion

Able to provide informed consent

Systolic blood pressure >125 mmHg

Impaired renal function defined as an eGFR of 30-75 mL/min/1.73 m2

Exclusion Criteria:

Use of other IV therapies for acute heart failure

Fever or sepsis

Recent major neurologic event

Recent major surgery

Recent acute coronary syndrome

Other recent investigational drug use

TABLE 4 Therapeutic Agents (TAs)-Nucleotide Sequences SEQ ID NAME NOSEQUENCE Oxyntomodulin 1 CACTCTCAGGGTACCTTCACCTCTGACTACTCTAAATACCTGGACTCTCGTCGTGCTCAGGACTTCGTTCAGTGGCTGATGAACACCAAACGTAACCGTAACAACATCGCT Relaxin 2GACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTTCGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTCTGGTGGCGGTCGTGGCGGTCGTCAGCTGTACTCTGCTCTGGCTAACAAATGCTGCCACGTTGGTTGCACC AAACGTTCTCTGGCTCGTTTCTGCTAARelaxin A Chain 3 CTGTACTCTGCTCTGGCTAACAAATGCTGCCACGTTGGTTGCACCAAACGTTCTCTGGCTCGTTTCTGC Relaxin SB29C 4GACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTTCGTGCTCAGATCGCTATCTGCGGTATGTCT ACCTGGTGC Leptin 5GTTCCGATCCAGAAAGTTCAGGACGACACCAAAACCCTGATCAAAACCATCGTTACCCGTATCAACGACATCTCTCACACCCAGTCTGTTTCTGCTAAACAGCGTGTTACtGGTCTGGACTTCATCCCGGGTCTGCACCCGATCCTGTCTCTGTCTAAAATGGACCAGACCCTGGCTGTTTACCAGCAGGTTCTGACCTCTCTGCCGTCTCAGAACGTTCTGCAGATCGCTAACGACCTGGAAAACCTGCGTGACCTGCTGCACCTGCTGGCTTTCTCTAAATCTTGCTCTCTGCCGCAGACCTCTGGTCTGCAGAAACCGGAATCTCTGGACGGTGTTCTGGAgGCTTCTCTGTACTCTACCGAAGTTGTTGCTCTGTCTCGTCTGCAGGGTTCTCTGCAGGACATCCTGCAGCAGCTGGACGTTTCTCC GGAATGCTAA Betatrophin 6GCTCCTCTGGGCGGTCCTGAACCAGCACAGTACGAGGAACTGACACTGTTGTTCCATGGAGCCTTGCAGCTGGGCCAGGCCCTCAACGGCGTGTACCGCGCCACAGAGGCACGTTTGACCGAGGCCGGACACAGCCTGGGTTTGTACGACAGAGCCCTGGAGTTTCTGGGTACCGAAGTGCGTCAGGGCCAGGACGCAACTCAGGAGCTGAGAACCTCCCTCTCTGAGATCCAGGTGGAGGAGGACGCCCTGCACCTGCGCGCCGAGGCGACAGCACGCTCTTTGGGAGAAGTTGCTCGCGCTCAGCAGGCCCTGCGTGATACCGTGCGGAGACTCCAAGTTCAGCTCAGAGGCGCTTGGCTCGGACAGGCGCATCAGGAGTTCGAGACCCTGAAAGCTCGTGCCGACAAACAGTCCCACCTGCTGTGGGCGCTCACCGGTCACGTCCAGCGCCAGCAACGCGAAATGGCCGAGCAGCAGCAATGGCTGCGCCAAATCCAG CAGCGCCTGCATACCGCGGCCCTGCCAGCGTAAFGF 21 7 CACCCGATCCCGGACTCTTCTCCGCTGCTGCAGTTCGGTGGTCAGGTTCGTCAGCGTTACCTGTACACCGACGACGCTCAGCAGACCGAAGCTCACCTGGAAATCCGTGAAGACGGTACtGTTGGTGGTGCTGCTGACCAGTCTCCGGAATCTCTGCTGCAGCTGAAAGCTCTGAAACCGGGTGTTATCCAGATCCTGGGTGTTAAAACCTCTCGTTTCCTGTGCCAGCGTCCGGACGGTGCTCTGTACGGTTCTCTGCACTTCGACCCGGAGGCATGCTCTTTCCGTGAACGTCTGCTGGAAGACGGTTACAACGTTTACCAGTCTGAAGCTCACGGTCTGCCGCTGCACCTGCCGGGTAACAAATCTCCGCACCGTGACCCGGCTCCGCGTGGTCCGGCTCGTTTCCTGCCGCTGCCGGGTCTGCCGCCGGCTCTGCCGGAACCGCCGGGTATCCTGGCTCCGCAGCCGCCGGACGTTGGTTCTTCTGACCCGCTGTCTATGGTTGGTGGTTCTCAGGGTCGTTCTCCGTCTTACGAATCTCCGT AA GDF 11 8AACCTGGGTCTGGACTGCGACGAACACTCTTCTGAATCTCGTTGCTGCCGTTACCCGCTGACCGTTGACTTCGAGGCGTTCGGTTGGGACTGGATCATCGCTCCGAAACGTTACAAAGCTAACTACTGCTCTGGTCAGTGCGAATACATGTTCATGCAGAAATACCCGCACACCCACCTGGTTCAGCAGGCTAACCCGCGTGGTTCTGCTGGTCCGTGCTGCACCCCGACCAAAATGTCTCCGATCAACATGCTGTACTTCAACGACAAACAGCAGATCATCTACGGTAAAATCCCGGGTATGGTTGTTGA CCGTTGCGGTTGCTCTTAA ANGPTL3 9GGATCCGGTGGTTTCACCATCAAACTGCTGCTGTTCATCGTTCCGCTGGTTATCTCTTCTCGTATCGACCAGGACAACTCTTCTTTCGACTCTCTGTCTCCGGAACCGAAATCTCGTTTCGCTATGCTGGACGACGTTAAAATCCTGGCTAACGGTCTGCTGCAGCTGGGTCACGGTCTGAAAGACTTCGTTCACAAAACCAAAGGTCAGATCAACGACATCTTCCAGAAACTGAACATCTTCGACCAGTCTTTCTACGACCTGTCTCTGCAGACCTCTGAAATCAAAGAAGAAGAAAAAGAACTGCGTCGTACCACCTACAAACTGCAGGTTAAAAACGAAGAAGTTAAAAACATGTCTCTGGAACTGAACTCTAAACTGGAATCTCTGCTGGAAGAAAAAATCCTGCTGCAGCAGAAAGTTAAATACCTGGAAGAACAGCTGACCAACCTGATCCAGAACCAGCCGGAAACCCCGGAACACCCGGAAGTTACCTCTCTGAAAACCTTCGTTGAAAAACAGGACAACTCTATCAAAGACCTGCTGCAGACCGTTGAAGACCAGTACAAACAGCTGAACCAGCAGCACTCTCAGATCAAAGAAATCGAAAACCAGCTGCGTCGTACCTCTATCCAGGAACCGACCGAAATCTCTCTGTCTTCTAAACCGCGTGCTCCGCGTACCACCCCGTTCCTGCAGCTGAACGAAATCCGTAACGTTAAACACGACGGTATCCCGGCTGAATGCACCACCATCTACAACCGTGGTGAACACACCTCTGGTATGTACGCTATCCGTCCGTCTAACTCTCAGGTTTTCCACGTTTACTGCGACGTTATCTCTGGTTCTCCGTGGACCCTGATCCAGCACCGTATCGACGGTTCTCAGAACTTCAACGAAACCTGGGAAAACTACAAATACGGTTTCGGTCGTCTGGACGGTGAATTCTGGCTGGGTCTGGAAAAAATCTACTCTATCGTTAAACAGTCTAACTACGTTCTGCGTATCGAACTGGAAGACTGGAAAGACAACAAACACTACATCGAATACTCTTTCTACCTGGGTAACCACGAAACCAACTACACCCTGCACCTGGTTGCTATCACCGGTAACGTTCCGAACGCTATCCCGAAGAAGAAGAAGAAAAAAAAGAAGAAGAA AT

TABLE 5 Therapeutic Agents-Amino acid sequences NAME SEQ ID NO SEQUENCEOxyntomodulin 10 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA Exendin-4 or 11HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Exenatide hGLP-1 12HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR Glucagon 13 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTRelaxin 14 DSWMEEVIKLCGRELVRAQIAICGMSTWSGGGRGGRQLY SALANKCCHVGCTKRSLARFCRelaxin A 15 LYSALANKCCHVGCTKRSLARFC Chain Relaxin SB29C 16DSWMEEVIKLCGRELVRAQIAICGMSTWC Leptin 17VPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEV VALSRLQGSLQDILQQLDVSPECBetatrophin 18 APLGGPEPAQYEELTLLFHGALQLGQALNGVYRATEARLTEAGHSLGLYDRALEFLGTEVRQGQDATQELRTSLSEIQVEEDALHLRAEATARSLGEVARAQQALRDTVRRLQVQLRGAWLGQAHQEFETLKARADKQSHLLWALTGHVQRQQRE MAEQQQWLRQIQQRLHTAALPA FGF 21 19HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGS SDPLSMVGGSQGRSPSYESP GDF 1120 NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTK MSPINMLYFNDKQQIIYGKIPGMVVDRCGCSANGPTL3 21 GSGGFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAI TGNVPNAIPKKKKKKKKKK Moka 22INVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYS VM-24 23AAAISCVGSPECPPKCRAQGCKNGKCMNRKCKCYYC Oxm-Cys38 24HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC Ex4-Cys40 25HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC Toxin H8-Tev- 26HHHHHHHHENLYFQGSCGGECIGMFKSCDPENDKCCKGR 550 TCSRKHRWCKYKL 550-GSGG 27GSGGEECIGMFKSCDPENDKCCKGRTCSRKHRWCKYKL (“GSGG” disclosed as SEQ ID NO:71) 550-4-GSCGG 28 GSCGGECIGMFKSCDPENDKCCKGRTCSRKHRWCKYKL (“GSCGG”disclosed as SEQ ID NO: 72) 550-3-GSGG 29GSGGECIGMFKSCDPENDKCCKGRTCSRKHRWCKYKLC (“GSGG” disclosed as SEQ ID NO:71) Toxin-550 30 ECIGMFKSCDPENDKCCKGRTCSRKHRWCKYKL 550-3 31ECIGMFKSCDPENDKCCKGRTCSRKHRWCKYKLGGSC Tev-Relaxin- 32*HHHHHHHHENLYFQGSGGDSWMEEVIKLCGRELVRAQI single-chainAICGMSTWSGGGRGGRQLYSALANKCCHVGCTKRSLARF C* GSGG-Relaxin- 33*GSGGDSWMEEVIKLCGRELVRAQIAICGMSTWSGGG single chainRGGRQLYSALANKCCHVGCTKRSLARFC* (“GSGG” disclosed as SEQ ID NO: 71)Tev-Relaxin-C- 34 *HHHHHHHHENLYFQGSGGDSWMEEVIKLCGRELVRA chainQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLY SALANKCCHVGCTKRSLARFC*Tev-Relaxin 35 *HHHHHHHHENLYFQSGGDSWMEEVIKLCGRELVRAQI Chain 1 AICGMSTWS*Tev-Relaxin 36 *QLYSALANKCCHVGCTKRSLARFC* Chain 2 Tev-Relaxin-B- 37*HHHHHHHHENLYFQGSGGDSWMEEVIKLCGRELVRAQI S29C-single-AICGMSTWCGGGRGGRQLYSALANKCCHVGCTKRSLAR chain FC* Tev-Relaxin-B- 38*HHHHHHHHENLYFQGSGGDSWMEEVIKLCGRELVRA S29C-C-chainQIAICGMSTWCKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLY SALANKCCHVGCTKRSLARFC*Pro-relaxin-B- 39 *MIEEGRDSWMEEVIKLCGRELVRAQIAICGMSTWSKRKP D1ATGYGSRKKRQLYSALANKCCHVGCTKRSLARFC* B-chain 40DSWMEEVIKLCGRELVRAQIAICGMSTWS A-chain 41 LYSALANKCCHVGCTKRSLARFCRelaxin A chain 1 42 QLYSALANKCCHVGCTKRSLARFC Relaxin A chain 2 43QLYSCLANKCCHVGCTKRSLARFC Relaxin A chain 3 44 QLYSALANKCCKVGCTKRSLARFCRelaxin A chain 4 45 CLYSALANKCCHVGCTKRSLARFC Relaxin A chain 5 46ALYSALANKCCAVGCTKRSLARFC Relaxin A chain 6 47 ALYSALANKCCAVGCTKRSLARFCRelaxin B chain 1 48 ASWMEEVIKLCGRELVRAQIAICGMSTWS Relaxin B chain 2 49ASWMEEVIKLCGRELVRAQIAICGMSTWC Relaxin B chain 3 50ASWKEEVIKLCGRELVRAQIAICGKSTWC Relaxin B chain 4 51ASWKEEVIKLCGRELVRAQIAICGMSTWC Relaxin B chain 5 52CSWMEEVIKLCGRELVRAQIAICGMSTWS H2-Relaxin A 53 ZLYSALANKCCHVGCTKRSLARFCchain H2-Relaxin B 54 DSWMEEVIKCLGRELVRAQIAICGMSTWS chainRelaxin A chain 7 55 QLYSALANKCCHVGCTKCSLARFC Relaxin B chain 6 56ASWMEEVIKLCGRELVRAQIAICGKSTWC Lowercase letters represent D-amino acidsTev = Tev protease cleavage sequence (ENLYFQ) (SEQ ID NO: 73) *= freeN-terminal or unmodified C-terminal carboxyl group

TABLE 6 Relaxin2 Construction Primer Sequences NAME SEQ ID NO SEQUENCERelaxin2 f1 57 TATTTCCAGGGATCCGGTGGTGACTCTTGGATGGAAGA (#32)AGTTATCAAACTGTGCGGTCGT Relaxin2 r6 58TAGACATACCGCAGATAGCGATCTGAGCACGAACCAG linker S26CTTCACGACCGCACAGTTTGATAA (#212) Relaxin2 Linker 59CGCTATCTGCGGTATGTCTACCTGGTCTGGTGGCGGTC f1 S26C (#213)GTGGCGGTCGTCAGCTGTACTC Relaxin2 Linker 60CGCTATCTGCGGTATGTCTACCTGGTCTGGTGGCGGTC f1 (#209) GTGGCGGTCGTCAGCTGTACTCRelaxin2 r6 61 TAGACATACCGCAGATAGCGATCTGAGCACGAACCAG (#43)TTCACGACCGCACAGTTTGATAA Relaxin2 Linker 62CGCTATCTGCGGTATGTCTACCTGGTCTGGTGGCGGTC f1 S29C (#214)GTGGCGGTCGTCAGCTGTACTC Relaxin2 Linker 63TTTGGTGCAACCAACGTGGCAGCATTTGTTAGCCAGAG r1 (#210) CAGAGTACAGCTGACGACCGCCpVB008 64 AATCTGTATTTCCAGGGATCCGGTGGTGA Relaxin2 Amp for (#60)Relaxin2 Linker 65 TGGCTAAGCTTTAGCAGAAACGAGCCAGAGAACGTTT Amp rev (#211)GGTGCAACCAACGTGGC

TABLE 7 XTEN-Amino acid sequences SEQ ID NAME NO SEQUENCE XTEN 66SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE 288TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR XTEN 67SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG 864SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG SETPGTSESATPESGPGTESASR

What is claimed is:
 1. A lipid conjugate (LC), comprising a. one or more lipids, the lipids selected from a group consisting of sterols, bile acids, vitamin E, fatty di-acids, fatty acids, fatty amides, fatty amines, fatty alcohols, and derivatives thereof; and b. a therapeutic agent (TA) comprising two amino acid sequences that are SEQ ID NO: 45 and 48; wherein the one or more lipids are conjugated to the TA via a cysteine residue at the N-terminus of SEQ ID NO:
 45. 2. The LC of claim 1, wherein the LC has the structure: TA-A¹-P¹-L  Formula (I) wherein: TA is the therapeutic agent; A¹ is a chemical group linking TA and P¹ or L; P¹ is a bond or comprises polyglycol; and L is the lipid.
 3. The LC of claim 2, wherein a sulfur atom of the cysteine residue of the TA is connected to A¹ via a chemical bond.
 4. The LC of claim 2, wherein A¹ is selected from

P¹ is -PEG-A²-; PEG is selected from

A² is selected from a bond,

L is tetradecanoic acid, octadecanedioic acid, tetradecylamine, myristic acid, stearic acid, docosahexaenoic acid, lithocholic acid ester, cholic acid or palmitic acid; X is a bond, NR⁵, S, or 0; each R¹, R², R³, and R⁴ is independently selected from H, halo, CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵; each R⁵ is independently H, alkyl, haloalkyl, arylalkyl, or heteroalkyl; R⁶ is OH or —NR⁵R⁵; each R⁷ is independently selected from H, alkyl, haloalkyl, arylalkyl, and heteroalkyl; m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; s is 1, 2, 3, 4, or 5; k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and q is 2, 3, 4, 5, 6, 7, 8, 9, or
 10. 5. The LC of claim 1, wherein the LC is selected from the following:


6. A pharmaceutical composition comprising the LC of claim
 1. 7. A method for treating a disease or condition selected from acute heart failure, congestive heart failure, compensated heart failure, decompensated heart failure, scleroderma, diffuse scleroderma, systemic scleroderma, fibromyalgia, fibrosis, and preeclampsia; or inducing labor in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of the LC of claim
 1. 8. The method of claim 7, wherein the LC is administered with one or more additional therapeutic agents selected from a group consisting of an anti-inflammatory drug, a statin, a diuretic, a beta-blocker, an angiotensin converting enzyme inhibitor, an angiotensin II receptor blocker or any combination thereof. 